ASIP-related proteins

ABSTRACT

The invention provides cDNAs which encode ASIP-related proteins. It also provides for the use of the cDNAs, fragments, complements, and variants thereof and of the encoded proteins, portions thereof and antibodies thereto for diagnosis and treatment of cancer, particularly bladder transitional cell carcinoma. The invention additionally provides expression vectors and host cells for the production of the protein and a transgenic model system.

FIELD OF THE INVENTION

[0001] This invention relates to cDNAs which encode ASIP-related proteins and to the use of the cDNAs and the encoded proteins in the diagnosis and treatment of cancer, particularly bladder transitional cell carcinoma.

BACKGROUND OF THE INVENTION

[0002] Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of molecules, biochemical and physiological mechanisms, and metabolic pathways. Despite different evolutionary pressures, the proteins of nematode, fly, rat, and man have common chemical and structural features and generally perform the same cellular function. Comparisons of the nucleic acid and protein sequences from organisms where structure and/or function are known accelerate the investigation of human sequences and allow the development of model systems for testing diagnostic and therapeutic agents for human conditions, diseases, and disorders.

[0003] Protein kinase C (PKC) plays roles in intracellular signaling through lipid-derived second messengers (Nishizuka (1995) FASEB J 9:484-496). Different isoforms of PKC have distinct tissue and subcellular distributions, show differential responses to lipids and calcium, and may serve different physiological functions. The PKC isoforms are divided into three classes, conventional PKC, novel PKC, and atypical PKC. The atypical PKC (aPKC) isoforms have been shown to play roles in Xenopus oocyte maturation (Dominguez et al. (1992) Mol Cell Biol 12:3776-3783), proliferation and survival of fibroblasts (Berra et al. (1993) Cell 74:555-563), differentiation of PC12 and leukemic cells (Wooten et al. (1994) Cell Growth Differ 5:395-403; Ways et al. (1994) Cell Growth Differ 5:1195-1203), activation of mitogen-activated protein kinase and gene expression (Berra et al. (1995) EMBO J 14:6157-6163; Lozano et al. (1994) J Biol Chem 269:19200-19202), insulin-induced glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane (Kotani et al. (1998) Mol Cell Biol 18:6971-6982), and the establishment and/or maintenance of cell polarity (Brazil and Hemmings (2000) Curr Biol 10:R592-594). Regulation by aPKC involves protein-protein interactions with other signaling molecules and aPKCs are known to interact in a number of different protein complexes (Moscat and Diaz-Meco (2000) EMBO Reports 1:399-403). The recruitment of aPKC into different complexes involves specific scaffold or adaptor proteins such as MyD88, TRADD, and p62.

[0004] One such adaptor protein may be atypical protein kinase C isotype specific interacting protein (ASIP), which binds to aPKCs including PKCζ and PKCλ (Izumi et al. (1998) J Cell Biol 143:95-106). Rat ASIP is 1337 amino acids in length and contains 3 PDZ domains that may mediate protein-protein interactions involved in the assembly of signaling complexes. The region of rat ASIP from amino acids 712-936 contains the binding site for aPKC. The sequence of ASIP is similar to that of the Caenorhabditis elegans protein, PAR-3, which is required for asymmetrical cell division in worm embryos. ASIP may also have a role in establishing and maintaining cell polarity in mammalian cells. Immunofluorescence microscopy studies show that ASIP and aPKCs colocalize at tight junctions and adherens junctions in epithelial cells where they may be involved in the formation and function of junctional complexes. Tight junctions maintain cellular asymmetry by providing a barrier to intramembrane diffusion between apical and basolateral membrane components as well as to diffusion between cells in an epithelial sheet. In addition, ASIP may have other roles in cellular signaling involving aPKC. For example, overexpression of ASIP in adiopocytes inhibits insulin stimulation of glucose uptake and translocation of the glucose transporter GLUT4 (Kotani et al. (2000) J Biol Chem 275:26390-26395). In the presence of excess ASIP, the amounts of PKCλ decrease in the cytosolic fraction and increase in the low density microsome and plasma membrane fractions. ASIP perturbs the subcellular distribution of PKCλ and interferes with the function of PKCλ in glucose uptake, possibly because of its direct interaction with PKCλ.

[0005] Regulation of cell proliferation by aPKC involves epidermal growth factor (EGF). EGF promotes proliferation and differentiation of mesenchymal and epithelial cells. It is a mitogen for fibroblasts, epithelial and endothelial cells, induces epithelial development, and promotes angiogenesis (Kim et al. (1999) Histol Histopathol 14:1175-1182 and Putz et al. (1999) Cancer Res 59:227-233). EGF is highly expressed in breast carcinoma cells and promotes tumor progression (Artagaveytia et al. (1997) J Steroid Biochem Mol Biol 60:221-228). EGF binds to its receptor, the protein tyrosine kinase EGF receptor (EGFR), which is also known as erbB2 (Carpenter (2000) Bioessays 22:697-707). Ligation of EGF to EGFR results in the activation of the tyrosine kinase domain of EGFR and the phosphorylation of multiple substrates. EGF may regulate multimeric signaling complexes associated with aPKCs. In cells stimulated with EGF, PKCλ is activated, shows an increased level of phosphorylation, and increased concentrations in the cytosol (Akimoto et al. (1996) EMBO J 15:788-798). EGF also influences the activity of PKCζ. In response to EGF, PKCζ phosphorylates and activates p70 S6 kinase, a regulator of cell proliferation (Romanelli et al. (1999) Mol Cell Biol 19:2921-2928).

[0006] The discovery of cDNAs encoding ASIP-related proteins satisfies a need in the art by providing compositions which are useful in the diagnosis and treatment of cancer, particularly bladder transitional cell carcinoma.

SUMMARY OF THE INVENTION

[0007] The invention is based on the discovery of cDNAs which encode ASIP-related proteins (ARP) which are useful in the diagnosis and treatment of cancer, particularly bladder transitional cell carcinoma.

[0008] The invention provides an isolated cDNA or a fragment thereof encoding a protein or a portion thereof selected from the group consisting of amino acid sequences of SEQ ID NO:1 (ARP-1) and SEQ ID NO:2 (ARP-2), a variant having at least at least 95% identity to the amino acid sequences of SEQ ID NO:1 or SEQ ID NO:2, an antigenic epitope of SEQ ID NO:1 or SEQ ID NO:2, and a biologically active portion of SEQ ID NO:1 or SEQ ID NO:2. The invention also provides an isolated cDNA or the complement thereof selected from the group consisting of a nucleic acid sequence of SEQ ID NO:3 and SEQ ID NO:20, a fragment of SEQ ID NO:3 selected from SEQ ID NOs:4-11 or a fragment of SEQ ID NO:20 selected from SEQ ID NOs:21-39, and a variant of SEQ ID NO:3 selected from SEQ ID NOs:12-19 or a variant of SEQ ID NO:20 selected from SEQ ID NOs:40-56. The invention additionally provides a composition, a substrate, and a probe comprising the cDNA, or the complement of the cDNA, encoding ARP-1 or ARP-2. The invention further provides a vector containing the cDNA, a host cell containing the vector and a method for using the cDNA to make ARP-1 or ARP-2. The invention still further provides a transgenic cell line or organism comprising the vector containing the cDNA encoding ARP. The invention additionally provides a fragment or the complement thereof selected from the group consisting of SEQ ID NOs:3-56. In one aspect, the invention provides a substrate containing at least one of these fragments. In a second aspect, the invention provides a probe comprising the fragment which can be used in methods of detection, screening, and purification. In a further aspect, the probe is a single stranded complementary RNA or DNA molecule.

[0009] The invention provides a method for using a cDNA to detect the differential expression of a nucleic acid in a sample comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the cDNA in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the cDNA is used to diagnose cancer, particularly bladder transitional cell carcinoma. In another aspect, the cDNA or a fragment or a complement thereof may comprise an element on an array.

[0010] The invention additionally provides a method for using a cDNA or a fragment or a complement thereof to screen a library or plurality of molecules or compounds to identify at least one ligand which specifically binds the cDNA, the method comprising combining the cDNA with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the cDNA, thereby identifying a ligand which specifically binds the cDNA. In one aspect, the molecules or compounds are selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, transcription factors, repressors, and regulatory molecules.

[0011] The invention provides a purified protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, a variant having at least 95% identity to the amino acid sequences of SEQ ID NO:1 or SEQ ID NO:2, an antigenic epitope of SEQ ID NO:1 or SEQ ID NO:2, and a biologically active portion of SEQ ID NO:1 or SEQ ID NO:2. The invention also provides a composition comprising the purified protein or a portion thereof in conjunction with a pharmaceutical carrier. The invention further provides a method of using the ARP-1 or ARP-2 to treat a subject with cancer, particularly bladder transitional cell carcinoma comprising administering to a patient in need of such treatment the composition containing the purified protein. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs. In another aspect, the ligand is used to treat a subject with cancer, particularly bladder transitional cell carcinoma.

[0012] The invention provides a method of using a protein to screen a subject sample for antibodies which specifically bind the protein comprising isolating antibodies from the subject sample, contacting the isolated antibodies with the protein under conditions that allow specific binding, dissociating the antibody from the bound-protein, and comparing the quantity of antibody with known standards, wherein the presence or quantity of antibody is diagnostic of cancer, particularly bladder transitional cell carcinoma.

[0013] The invention also provides a method of using a protein to prepare and purify antibodies comprising immunizing a animal with the protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified antibodies.

[0014] The invention provides a purified antibody which binds specifically to a protein which is expressed in cancer, particularly bladder transitional cell carcinoma. The invention also provides a method of using an antibody to diagnose cancer, particularly bladder transitional cell carcinoma comprising combining the antibody comparing the quantity of bound antibody to known standards, thereby establishing the presence of cancer, particularly bladder transitional cell carcinoma. The invention further provides a method of using an antibody to treat cancer, particularly bladder transitional cell carcinoma comprising administering to a patient in need of such treatment a pharmaceutical composition comprising the purified antibody.

[0015] The invention provides a method for inserting a marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide. The invention also provides a method for using a cDNA to produce a mammalian model system, the method comprising constructing a vector containing the cDNA selected from SEQ ID NOs:3-56, transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem, microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system.

BRIEF DESCRIPTION OF THE FIGURES AND TABLE

[0016]FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J and 1K show the ARP-1 (SEQ ID NO:1) encoded by the cDNA (SEQ ID NO:3). The translation was produced using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco Calif.).

[0017]FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2 H, 2I, 2J, 2K, 2L, 2M, 2N, 2Q, 2R, and 2S show the ARP-2 (SEQ ID NO:2) encoded by the cDNA (SEQ ID NO:20). The translation was produced using MACDNASIS PRO software (Hitachi Software Engineering).

[0018]FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I and 3J demonstrate the conserved chemical and structural similarities among the sequences and domains of ARP-1 (1555118; SEQ ID NO:1), ARP-2 (2582063; SEQ ID NO:2), rat ASIP (g3868778; SEQ ID NO:62), and human ASIP (g8037915; SEQ ID NO:63). The translation was produced using the MEGALIGN program of LASERGENE software (DNASTAR, Madison Wis.).

[0019] Tables 1 and 2 show the northern analysis for ARP-1 and ARP-2 produced using the LIFESEQ Gold database (Incyte Genomics, Palo Alto Calif.). In Table 1, the first column presents the tissue categories; the second column, the total number of clones in the tissue category; the third column, the ratio of the number of libraries in which at least one transcript was found to the total number of libraries; the fourth column, absolute clone abundance of the transcript; and the fifth column, percent abundance of the transcript. Table 2 shows expression of ARP in bladder tissue. The first column lists the library name, the second column, the number of clones sequenced for that library; the third column, the description of the tissue from which the library was derived; the fourth column, the absolute abundance of the transcript; and the fifth column, the percent abundance of the transcript.

[0020] Table 3 shows the differential expression of ARP-2 in human BT20 breast carcinoma cells treated with EGF compared to untreated cells as determined by microarray analysis. Column 1 lists the mean differential expression (DE) values presented as log2 DE (treated cells/untreated cells). Column 2 lists the untreated control samples labeled with fluorescent green dye Cy3. Column 3 lists the treatment for samples labeled with fluorescent red dye Cy5.

DESCRIPTION OF THE INVENTION

[0021] It is understood that this invention is not limited to the particular machines, materials and methods described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the scope of the present invention which will be limited only by the appended claims. As used herein, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. For example, a reference to “a host cell” includes a plurality of such host cells known to those skilled in the art.

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Definitions

[0023] “ARP” refers to a substantially purified protein obtained from any mammalian species, including bovine, canine, murine, ovine, porcine, rodent, simian, and preferably the human species, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.

[0024] “Array” refers to an ordered arrangement of at least two cDNAs on a substrate. At least one of the cDNAs represents a control or standard sequence, and the other, a cDNA of diagnostic interest. The arrangement of from about two to about 40,000 cDNAs on the substrate assures that the size and signal intensity of each labeled hybridization complex formed between a cDNA and a sample nucleic acid is individually distinguishable.

[0025] The “complement” of a cDNA of the Sequence Listing refers to a nucleic acid molecule which is completely complementary over its full length and which will hybridize to the cDNA or an mRNA under conditions of high stringency.

[0026] “cDNA” refers to an isolated polynucleotide, nucleic acid molecule, or any fragment or complement thereof. It may have originated recombinantly or synthetically, be double-stranded or single-stranded, represent coding and/or noncoding 5′ and 3′ sequence.

[0027] The phrase “cDNA encoding a protein” refers to a nucleic acid sequence that closely aligns with sequences which encode conserved regions, motifs or domains that were identified by employing analyses well known in the art. These analyses include BLAST (Basic Local Alignment Search Tool; Altschul (1993) J Mol Evol 36: 290-300; Altschul et al. (1990) J Mol Biol 215:403-410) which provides identity within the conserved region.

[0028] “Derivative” refers to a cDNA or a protein that has been subjected to a chemical modification. Derivatization of a cDNA can involve substitution of a nontraditional base such as queosine or of an analog such as hypoxanthine. These substitutions are well known in the art. Derivatization of a protein involves the replacement of a hydrogen by an acetyl, acyl, alkyl, amino, formyl, or morpholino group. Derivative molecules retain the biological activities of the naturally occurring molecules but may confer advantages such as longer lifespan or enhanced activity.

[0029] “Differential expression” refers to an increased, upregulated or present, or decreased, downregulated or absent, gene expression as detected by the absence, presence, or at least two-fold changes in the amount of transcribed messenger RNA or translated protein in a sample.

[0030] “Disorder” refers to conditions, diseases or syndromes in which the cDNAs and ARP are differentially expressed such as cancer, particularly bladder transitional cell carcinoma.

[0031] “Fragment” refers to a chain of consecutive nucleotides from about 200 to about 700 base pairs in length. Fragments may be used in PCR or hybridization technologies to identify related nucleic acid molecules and in binding assays to screen for a ligand. Nucleic acids and their ligands identified in this manner are useful as therapeutics to regulate replication, transcription or translation.

[0032] A “hybridization complex” is formed between a cDNA and a nucleic acid of a sample when the purines of one molecule hydrogen bond with the pyrimidines of the complementary molecule, e.g., 5′-A -G-T-C-3′ base pairs with 3′-T-C-A-G-5′. The degree of complementarity and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions.

[0033] “Ligand” refers to any agent, molecule, or compound which will bind specifically to a complementary site on a cDNA molecule or polynucleotide, or to an epitope or a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic or organic substances including nucleic acids, proteins, carbohydrates, fats, and lipids.

[0034] “Oligonucleotide” refers a single stranded molecule from about 18 to about 60 nucleotides in length which may be used in hybridization or amplification technologies or in regulation of replication, transcription or translation. Substantially equivalent terms are amplimer, primer, and oligomer.

[0035] “Portion” refers to any part of a protein used for any purpose; but especially, to an epitope for the screening of ligands or for the production of antibodies.

[0036] “Post-translational modification” of a protein can involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and the like. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cellular location, cell type, pH, enzymatic milieu, and the like.

[0037] “Probe” refers to a cDNA that hybridizes to at least one nucleic acid in a sample. Where targets are single stranded, probes are complementary single strands. Probes can be labeled with reporter molecules for use in hybridization reactions including Southern, northern, in situ, dot blot, array, and like technologies or in screening assays.

[0038] “Protein” refers to a polypeptide or any portion thereof. A “portion” of a protein refers to that length of amino acid sequence which would retain at least one biological activity, a domain identified by PFAM or PRINTS analysis or an antigenic epitope of the protein identified using Kyte-Doolittle algorithms of the PROTEAN program (DNASTAR, Madison Wis.). An “oligopeptide” is an amino acid sequence from about five residues to about 15 residues that is used as part of a fusion protein to produce an antibody.

[0039] “Purified” refers to any molecule or compound that is separated from its natural environment and is from about 60% free to about 90% free from other components with which it is naturally associated.

[0040] “Sample” is used in its broadest sense as containing nucleic acids, proteins, antibodies, and the like. A sample may comprise a bodily fluid; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, buccal cells, skin, or hair; and the like.

[0041] “Specific binding” refers to a special and precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule, the hydrogen bonding along the backbone between two single stranded nucleic acids, or the binding between an epitope of a protein and an agonist, antagonist, or antibody.

[0042] “Similarity” as applied to sequences, refers to the quantification (usually percentage) of nucleotide or residue matches between at least two sequences aligned using a standardized algorithm such as Smith-Waterman alignment (Smith and Waterman (1981) J Mol Biol 147:195-197) or BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-3402). BLAST2 may be used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them.

[0043] “Substrate” refers to any rigid or semi-rigid support to which cDNAs or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.

[0044] “Variant” refers to molecules that are recognized variations of a cDNA or a protein encoded by the cDNA. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to the cDNAs and may differ by about three bases per hundred bases. “Single nucleotide polymorphism” (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid or its secondary, tertiary, or quaternary structure.

The Invention

[0045] The invention is based on the discovery of a cDNA which encodes ARP and on the use of the cDNA, or fragments thereof, and protein, or portions thereof, directly or as compositions in the characterization, diagnosis, and treatment of cancer.

[0046] Nucleic acids encoding the ARP-1 of the present invention were first identified in Incyte Clone 1555118 from the human bladder tumor cDNA library (BLADTUT04) using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO;2, was derived from the following overlapping and/or extended nucleic acid sequences (SEQ ID NO:4-11): Incyte Clones 1555118H1 (BLADTUT04),7227391H1 (BRAXTDR15), 70158486V1 (SG0000039), 70162686V1 (SG0000040), 70151326V1 (SG0000038), 70154198V1 (SG0000038),2084238T6 (UTRSNOT08), 70155923V1 (SG0000038), and GenBank EST g6661750 (SEQ ID NO:57), and edited Genscan sequence GNN.g10801482_(—)004.edit (SEQ ID NO:58). For sequence GNN.g10801482_(—)004.edit, coding regions were predicted by Genscan analysis of the genomic DNA. g10801482 is the GenBank identification number of the sequence to which Genscan was applied.

[0047] In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:1 as shown in FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J and 1K. ARP-1 is 935 amino acids in length and has four potential N-glycosylation sites at N332, N502, N516, and N778; two indicates that the regions of ARP-1 from T203 to P290, K383 to Q469, and E498 to R591 are similar to PDZ domains. Such domains participate in protein-protein interactions with signaling molecules. As shown in FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I and 3J, ARP-1 has chemical and structural similarity with rat ASIP (g3868778; SEQ ID NO:62) and human ASIP (g8⁰37915; SEQ ID NO:63). In particular, ARP-1 and rat ASIP share about 41% identity and ARP-1 and human ASIP share about 43% identity. The region of ARP-1 from S708 to F925 is similar to the aPCK binding regions of rat ASIP and human ASIP. All three proteins share three conserved PDZ domains and potential protein kinase C phosphorylation sites. Useful antigenic epitopes of ARP-1 extend from R192 to S222, K413 to S455, and K759 to Q809; and biologically active portions of ARP-1 extend from T203 to P290, K383 to Q469, E498 to R591, and S708 to F925. An antibody which specifically binds ARP-1 is useful in a diagnostic assay for cancer, particularly bladder transitional cell carcinoma.

[0048] Nucleic acids encoding the ARP-2 of the present invention were first identified in Incyte Clone 2582063 from the human bladder tumor cDNA library (KIDNTUT13) using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO:2, was derived from the following overlapping and/or extended nucleic acid sequences (SEQ ID NO:4-1 1): Incyte Clones 2582063H1 (KIDNTUT13), 7246093H1 (PROSTMYO1), 7978420H1, 55040412H1, 2929484F6 (TLYMNOT04), 5627320R8 (PLACFER01), 3209128F6 (BLADNOT08), 349248H1 (LVENNOT01), 7019961H1 (PANCNON03), 6303175H2 (TLYMUNT02), 2549906F6 (LUNGTUT06), 1945452H1 (PITUNOT01), 2549906T6 (LUNGTUT06), 71009002V1 (SG0000308), 71008521V1 (SG0000308), 71010168V1 (SG0000308), 70090181V1 (SG0000030), 6833928H1 (BRSTNON02), 70089663V1 (SG0000030) and GenBank ESTs g6993427 (SEQ ID NO:59), g5529915 (SEQ ID NO:60), and g1733437 (SEQ ID NO:61).

[0049] In another embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:2 as shown in FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 21, 2J, 2k, 2L, 2M, 2N, 2O, 2P, 2Q, 2R and 2S. ARP-2 is 1356 amino acids in length and has ten potential N-glycosylation sites at N112, N159, N208, N265, N391, N594, N608, N922, N1144, and N1231; six potential cyclic AMP-or cyclic GMP-dependent protein kinase phosphorylation sites at S144, T468, S685, S715, S888, and T1176; thirty-four potential casein kinase II phosphorylation sites at S123, S154, S161, T243, S248, T258, S267, S370, T476, S523, T534, T569, T654, T705, S720, S780, S781, S827, S840, T865, S888, T947, S958, T965, T995, T1048, S1049, S1102, S1116, S1139, S1149, S1245, S1252, and S1308; twenty-six potential protein kinase C phosphorylation sites at S166, S187, S292, S372, S379, S428, T453, T476, S533, S604, T661, S742, S829, T847, S924, S955, S958, T988, T995, T1038, S1116, S1143, S1192, S1228, S1252, and S1312; six potential tyrosine kinase phosphorylation sites at Y199, Y388, Y745, Y933, Y1080, and Y1321; two potential ATP/GTP-binding sites motif A (P-loop) from G516 through S523 and from G647 through S654. PFAM analysis indicates that the regions of ARP-2 from K273 to A360, N461 to Q547, and E590 to R683 are similar to PDZ domains. As shown in FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I and 3J, ARP-2 has chemical and structural similarity with rat ASIP (g3868778; SEQ ID NO:62) and human ASIP (g8037915; SEQ ID NO:63). In particular, ARP-2 and rat ASIP share about 89% identity and ARP-2 and human ASIP share about 93% identity. The region of ARP-2 from R712 to K936 is similar to the aPCK binding regions of rat ASIP and human ASIP. All three proteins share three conserved PDZ domains and potential protein kinase C phosphorylation sites. Useful antigenic epitopes of ARP-2 extend from K189 to Q236, T895 to M1023, and R1207 to N1267; and biologically active portions of ARP-2 extend from K273 to A360, N461 to Q547, E590 to R683, and R712 to K936. An antibody which specifically binds ARP-2 is useful in a diagnostic assay for cancer, particularly bladder transitional cell carcinoma.

[0050] Table 1 shows expression of ARP-1 and ARP-2 across the tissue categories (also listed in Example VIII). Table 2 shows expression of ARP in bladder tissues, particularly in tissues from patients with transitional cell carcinoma. ARP shows overexpression in a library (BLADTUT04) from bladder tissue from a patient with transitional cell carcinoma compared to a library (BLADNOT05) from matched (m) microscopically normal tissue from the same donor. Table 3 shows the differential expression of ARP-2 in human BT20 breast carcinoma cells treated with EGF as determined by microarray analysis. ARP-2 shows reduced expression in BT20 cells treated with EGF for 4 to 48 hours compared to untreated cells. The largest decrease in expression in treated BT20 cells is observed at 36 hours. Therefore, the cDNAs encoding ARP-1 or ARP-2 are useful in assays to diagnose cancer, particularly bladder transitional cell carcinoma. Fragments of the cDNA encoding ARP-1 from about nucleotide 1792 to about nucleotide 1836, from about nucleotide 1905 to about nucleotide 1950, and from about nucleotide 2180 to about nucleotide 2230 are also useful in diagnostic assays. Fragments of the cDNA encoding ARP -2 from about nucleotide 585 to about nucleotide 635 and from about nucleotide 1670 to about nucleotide 1710 are also useful in diagnostic assays.

[0051] Mammalian variants of the cDNA encoding ARP were identified using BLAST2 with default parameters and the ZOOSEQ databases (Incyte Genomics). These preferred variants have from about 80% to about 100% identity as shown in the table below. The first column shows the SEQ ID for the human cDNA (SEQ ID_(H)); the second column, the SEQ ID for the variant cDNAs (SEQ ID_(var)); the third column, the clone number for the variant cDNAs (Clone_(var)); the fourth column, the library name; the fifth column, the alignment of the variant cDNA to the human cDNA (includes the alignment of different regions of the variant cDNA with different regions of the human cDNA in some cases); and the sixth

[0052] Mammalian variants of the cDNA encoding ARP were identified using BLAST2 with default parameters and the ZOOSEQ databases (Incyte Genomics). These preferred variants have from about 80% to about 100% identity as shown in the table below. The first column shows the SEQ ID for the human cDNA (SEQ ID_(H)); the second column, the SEQ ID for the variant cDNAs (SEQ ID_(var)); the third column, the clone number for the variant cDNAs (Clone_(var)); the fourth column, the library name; the fifth column, the alignment of the variant cDNA to the human cDNA (includes the alignment of different regions of the variant cDNA with different regions of the human cDNA in some cases); and the sixth column, the percent identity to the human cDNA. SEQ ID_(H) SEQ ID_(var) Clone_(var) Library Name Nt_(H) Alignment Identity 3 12 702457609T1 RACONON05 2162-2619 84% 3 13 702458746T1 RACONON05 2152-2579 84% 3 14 701335936H1 RALINON08 1322-1566 88% 3 15 700639694H1 RATONOT01 2361-2619 87% 3 16 700639694F6 RATONOT01 2447-2651 85% 3 17 701191467H1 RACONON05 2063-2333 81% 3 18 702771158H1 CNLINOT02 615-710 87% 3 19 701266650H1 MOLUDIT08 1289-1544 91% 20 40 702231139H1 RAFANOT02 2500-2951 88% 20 41 700273304F6 RASJNOT01 2705-3074 85% 20 42 700330856H1 RALINON04 1550-1817 87% 20 43 700273304H1 RASJNOT01 2705-2951 86% 20 44 701517518H1 RALITXT62 2149-2441 83% 20 45 701834089T1 RAKITXT10 2897-3074 85% 20 46 701480437H1 RALITXT42 3679-3824 88% 20 47 701190235H1 RACONON05 2077-2333 80% 20 48 700939688H1 RALINON07 2971-3074 88% 20 49 700939688F6 RALINON07 2971-3074 88% 20 50 702582937T1 RABYUNN01 5276-5319 95% 20 51 700299037F6 RAEONOT02 5276-5334 91% 20 52 701246488H1 RALINOH02 2284-2357 86% 20 53 702759912H1 CNLIUNN01   1-566 92% 20 54 700112340H1 MOOSUNR01 121-362 88% 20 55 700827810H1 MOEMNOT01 3559-3701 87% 20 56 700109331H1 MOOSUNR01 2509-2569 100%

[0053] These cDNAs are particularly useful for producing transgenic cell lines or organisms which model human disorders and upon which potential therapeutic treatments for such disorders may be tested.

[0054] It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of cDNA encoding ARP, some bearing minimal similarity to the cDNAs of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of cDNA that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide encoding naturally occurring ARP, and all such variations are to be considered as being specifically disclosed.

[0055] The cDNA and fragments thereof (SEQ ID NOs:3-56) may be used in hybridization, amplification, and screening technologies to identify and distinguish among SEQ ID NO:3, SEQ ID NO:20 and related molecules in a sample. The mammalian cDNAs may be used to produce transgenic cell lines or organisms which are model systems for human cancer, particularly bladder transitional cell carcinoma and upon which the toxicity and efficacy of potential therapeutic treatments may be tested. Toxicology studies, clinical trials, and subject/patient treatment profiles may be performed and monitored using the cDNAs, proteins, antibodies and molecules and compounds identified using the cDNAs and proteins of the present invention.

Characterization and Use of the Invention cDNA Libraries

[0056] In a particular embodiment disclosed herein, mRNA was isolated from mammalian cells and tissues using methods which are well known to those skilled in the art and used to prepare the cDNA libraries. The Incyte clones listed above were isolated from mammalian cDNA libraries. Three library preparations representative of the invention are described in the EXAMPLES below. The consensus sequences were chemically and/or electronically assembled from fragments including Incyte clones and extension and/or shotgun sequences using computer programs such as PHRAP (P Green, University of Washington, Seattle Wash.), and AUTOASSEMBLER application (Applied Biosystems, Foster City Calif.). Clones, extension and/or shotgun sequences are electronically assembled into clusters and/or master clusters.

Sequencing

[0057] Methods for sequencing nucleic acids are well known in the art and may be used to practice any of the embodiments of the invention. These methods employ enzymes such as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNA polymerase (Amersham Pharmacia Biotech (APB), Piscataway N.J.), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg Md.). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 system (Hamilton, Reno Nev.) and the DNA ENGINE thermal cycler (MJ Research, Watertown Mass.). Machines commonly used for sequencing include the ABI PRISM 3700, 377 or 373 DNA sequencing systems (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (APB), and the like. The sequences may be analyzed using a variety of algorithms well known in the art and described in Ausubel et al. (1997; Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., unit 7.7) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., pp. 856-853).

[0058] Shotgun sequencing may also be used to complete the sequence of a particular cloned insert of interest. Shotgun strategy involves randomly breaking the original insert into segments of various sizes and cloning these fragments into vectors. The fragments are sequenced and reassembled using overlapping ends until the entire sequence of the original insert is known. Shotgun sequencing methods are well known in the art and use thermostable DNA polymerases, heat-labile DNA polymerases, and primers chosen from representative regions flanking the cDNAs of interest. Incomplete assembled sequences are inspected for identity using various algorithms or programs such as CONSED (Gordon (1998) Genome Res 8:195-202) which are well known in the art. Contaminating sequences including vector or chimeric sequences or deleted sequences can be removed or restored, respectively, organizing the incomplete assembled sequences into finished sequences.

Extension of a Nucleic Acid Sequence

[0059] The sequences of the invention may be extended using various PCR-based methods known in the art. For example, the XL-PCR kit (Applied Biosystems), nested primers, and commercially available cDNA or genomic DNA libraries may be used to extend the nucleic acid sequence. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO primer analysis software (Molecular Biology Insights, Cascade Colo.) to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to a target molecule at temperatures from about 55C. to about 68C. When extending a sequence to recover regulatory elements, it is preferable to use genomic, rather than cDNA libraries.

Hybridization

[0060] The cDNA and fragments thereof can be used in hybridization technologies for various purposes. A probe may be designed or derived from unique regions such as the 5′ regulatory region or from a nonconserved region (i.e., 5′ or 3′ of the nucleotides encoding the conserved catalytic domain of the protein) and used in protocols to identify naturally occurring molecules encoding the ARP, allelic variants, or related molecules. The probe may be DNA or RNA, may be single stranded and should have at least 50% sequence identity to any of the nucleic acid sequences, SEQ ID NOs:3-56. Hybridization probes may be produced using oligolabeling, nick translation, end-labeling, or PCR amplification in the presence of a reporter molecule. A vector containing the cDNA or a fragment thereof may be used to produce an mRNA probe in vitro by addition of an RNA polymerase and labeled nucleotides. These procedures may be conducted using commercially available kits such as those provided by APB.

[0061] The stringency of hybridization is determined by G+C content of the probe, salt concentration, and temperature. In particular, stringency can be increased by reducing the concentration of salt or raising the hybridization temperature. In solutions used for some membrane based hybridizations, addition of an organic solvent such as formamide allows the reaction to occur at a lower temperature. Hybridization can be performed at low stringency with buffers, such as 5×SSC with 1% sodium dodecyl sulfate (SDS) at 60C., which permits the formation of a hybridization complex between nucleic acid sequences that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2×SSC with 0.1% SDS at either 45C. (medium stringency) or 68C. (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acids are completely complementary. In some membrane-based hybridizations, preferably 35% or most preferably 50%, formamide can be added to the hybridization solution to reduce the temperature at which hybridization is performed, and background signals can be reduced by the use of other detergents such as Sarkosyl or TRITON X-100 (Sigma-Aldrich, St. Louis Mo.) and a blocking agent such as denatured salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel (supra) and Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.

[0062] Arrays may be prepared and analyzed using methods known in the art. Oligonucleotides may be used as either probes or targets in an array. The array can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and single nucleotide polymorphisms. Such information may be used to determine gene function; to understand the genetic basis of a condition, disease, or disorder; to diagnose a condition, disease, or disorder; and to develop and monitor the activities of therapeutic agents. (See, e.g., Brennan et al. (1995) USPN 5,474,796; Schena et al. (1996) Proc Natl Acad Sci 93:10614-10619; Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon et al. (1995) PCT application WO95/35505; Heller et al. (1997) Proc Natl Acad Sci 94:2150-2155; and Heller et al. (1997) USPN 5,605,662.)

[0063] Hybridization probes are also useful in mapping the naturally occurring genomic sequence. The probes may be hybridized to: 1) a particular chromosome, 2) a specific region of a chromosome, or 3) an artificial chromosome construction such as human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), bacterial P1 construction, or single chromosome cDNA libraries.

Expression

[0064] Any one of a multitude of cDNAs encoding ARP may be cloned into a vector and used to express the protein, or portions thereof, in host cells. The nucleic acid sequence can be engineered by such methods as DNA shuffling (U.S. Pat No. 5,830,721) and site-directed mutagenesis to create new restriction sites, alter glycosylation patterns, change codon preference to increase expression in a particular host, produce splice variants, extend half-life, and the like. The expression vector may contain transcriptional and translational control elements (promoters, enhancers, specific initiation signals, and polyadenylated 3′ sequence) from various sources which have been selected for their efficiency in a particular host. The vector, cDNA, and regulatory elements are combined using in vitro recombinant DNA techniques, synthetic techniques, and/or in vivo genetic recombination techniques well known in the art and described in Sambrook (supra, ch. 4, 8, 16 and 17).

[0065] A variety of host systems may be transformed with an expression vector. These include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems transformed with baculovirus expression vectors; plant cell systems transformed with expression vectors containing viral and/or bacterial elements, or animal cell systems (Ausubel supra, unit 16). For example, an adenovirus transcription/translation complex may be utilized in mammalian cells. After sequences are ligated into the E1 or E3 region of the viral genome, the infective virus is used to transform and express the protein in host cells. The Rous sarcoma virus enhancer or SV40 or EBV-based vectors may also be used for high-level protein expression.

[0066] Routine cloning, subcloning, and propagation of nucleic acid sequences can be achieved using the multifunctional PBLUESCRIPT vector (Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Life Technologies). Introduction of a nucleic acid sequence into the multiple cloning site of these vectors disrupts the lacZ gene and allows colorimetric screening for transformed bacteria. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.

[0067] For long term production of recombinant proteins, the vector can be stably transformed into cell lines along with a selectable or visible marker gene on the same or on a separate vector. After transformation, cells are allowed to grow for about 1 to 2 days in enriched media and then are transferred to selective media. Selectable markers, antimetabolite, antibiotic, or herbicide resistance genes, confer resistance to the relevant selective agent and allow growth and recovery of cells which successfully express the introduced sequences. Resistant clones identified either by survival on selective media or by the expression of visible markers, such as anthocyanins, green fluorescent protein (GFP), B glucuronidase, luciferase and the like, may be propagated using culture techniques. Visible markers are also used to quantify the amount of protein expressed by the introduced genes. Verification that the host cell contains the desired cDNA is based on DNA-DNA or DNA-RNA hybridizations or PCR amplification techniques.

[0068] The host cell may be chosen for its ability to modify a recombinant protein in a desired fashion. Such modifications include acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation and the like. Post-translational processing which cleaves a “prepro” form may also be used to specify protein targeting, folding, and/or activity. Different host cells available from the ATCC (Manassas Va.) which have specific cellular machinery and characteristic mechanisms for post-translational activities may be chosen to ensure the correct modification and processing of the recombinant protein.

Recovery of Proteins from Cell Culture

[0069] Heterologous moieties engineered into a vector for ease of purification include glutathione S-transferase (GST), 6×His, FLAG, MYC, and the like. GST and 6-His are purified using commercially available affinity matrices such as immobilized glutathione and metal-chelate resins, respectively. FLAG and MYC are purified using commercially available monoclonal and polyclonal antibodies. For ease of separation following purification, a sequence encoding a proteolytic cleavage site may be part of the vector located between the protein and the heterologous moiety. Methods for recombinant protein expression and purification are discussed in Ausubel (supra, unit 16) and are commercially available.

Chemical Synthesis of Peptides

[0070] Proteins or portions thereof may be produced not only by recombinant methods, but also by using chemical methods well known in the art. Solid phase peptide synthesis may be carried out in a batchwise or continuous flow process which sequentially adds a-amino- and side chain-protected amino acid residues to an insoluble polymeric support via a linker group. A linker group such as methylamine-derivatized polyethylene glycol is attached to poly(styrene-co-divinylbenzene) to form the support resin. The amino acid residues are N-α-protected by acid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc (9-fluorenylmethoxycarbonyl). The carboxyl group of the protected amino acid is coupled to the amine of the linker group to anchor the residue to the solid phase support resin. Trifluoroacetic acid or piperidine are used to remove the protecting group in the case of Boc or Fmoc, respectively. Each additional amino acid is added to the anchored residue using a coupling agent or pre-activated amino acid derivative, and the resin is washed. The full length peptide is synthesized by sequential deprotection, coupling of derivitized amino acids, and washing with dichloromethane and/or N, N-dimethylformamide. The peptide is cleaved between the peptide carboxy terminus and the linker group to yield a peptide acid or amide. (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook, San Diego Calif. pp. S1-S20). Automated synthesis may also be carried out on machines such as the ABI 431 A peptide synthesizer (Applied Biosystems). A protein or portion thereof may be substantially purified by preparative high performance liquid chromatography and its composition confirmed by amino acid analysis or by sequencing (Creighton (1984) Proteins, Structures and Molecular Properties, W H Freeman, New York N.Y.).

Preparation and Screening of Antibodies

[0071] Various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with ARP or any portion thereof. Adjuvants such as Freund's, mineral gels, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemacyanin (KLH), and dinitrophenol may be used to increase immunological response. The oligopeptide, peptide, or portion of protein used to induce antibodies should consist of at least about five amino acids, more preferably ten amino acids, which are identical to a portion of the natural protein. Oligopeptides may be fused with proteins such as KLH in order to produce antibodies to the chimeric molecule.

[0072] Monoclonal antibodies may be prepared using any technique which provides for the production of antibodies by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler et al. (1975) Nature 256:495-497; Kozbor et al. (1985) J. Immunol Methods 81:31-42; Cote et al. (1983Proc Natl Acad Sci 80:2026-2030; and Cole et al. (1984) Mol Cell Biol 62:109-120.)

[0073] Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce epitope specific single chain antibodies. Antibody fragments which contain specific binding sites for epitopes of the protein may also be generated. For example, such fragments include, but are not limited to, F(ab′)2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse et al. (1989) Science 246:1275-1281.)

[0074] The ARP or a portion thereof may be used in screening assays of phagemid or B-lymphocyte immunoglobulin libraries to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoassays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may also be employed (Pound (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).

Labeling of Molecules for Assay

[0075] A wide variety of reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid, amino acid, and antibody assays. Synthesis of labeled molecules may be achieved using commercially available kits (Promega, Madison Wis.) for incorporation of a labeled nucleotide such as ³²P-dCTP (APB), Cy3-dCTP or Cy5-dCTP (Operon Technologies, Alameda Calif.), or amino acid such as ³⁵S-methionine (APB). Nucleotides and amino acids may be directly labeled with a variety of substances including fluorescent, chemiluminescent, or chromogenic agents, and the like, by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FITC (Molecular Probes, Eugene Oreg.).

Diagnostics

[0076] The cDNAs, fragments, oligonucleotides, complementary RNA and DNA molecules, and PNAs and may be used to detect and quantify differential gene expression, absence/presence vs. excess, expression of mRNAs or to monitor mRNA levels during therapeutic intervention. Similarly antibodies which specifically bind ARP may be used to quantitate the protein. Disorders associated with differential expression include cancer, particularly bladder transitional cell carcinoma. The diagnostic assay may use hybridization or amplification technology to compare gene expression in a biological sample from a patient to standard samples in order to detect differential gene expression. Qualitative or quantitative methods for this comparison are well known in the art.

[0077] For example, the cDNA or probe may be labeled by standard methods and added to a biological sample from a patient under conditions for the formation of hybridization complexes. After an incubation period, the sample is washed and the amount of label (or signal) associated with hybridization complexes, is quantified and compared with a standard value. if complex formation in the patient sample is significantly altered (higher or lower) in comparison to either a normal or disease standard, then differential expression indicates the presence of a disorder.

[0078] In order to provide standards for establishing differential expression, normal and disease expression profiles are established. This is accomplished by combining a sample taken from normal subjects, either animal or human, with a cDNA under conditions for hybridization to occur. Standard hybridization complexes may be quantified by comparing the values obtained using normal subjects with values from an experiment in which a known amount of a substantially purified sequence is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who were diagnosed with a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular disorder is used to diagnose that disorder.

[0079] Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies and in clinical trial or to monitor the treatment of an individual patient. Once the presence of a condition is established and a treatment protocol is initiated, diagnostic assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.

Immunological Methods

[0080] Detection and quantification of a protein using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may be employed. (See, e.g., Coligan et al. (1997) Current Protocols in Immunology, Wiley-Interscience, New York N.Y.; and Pound, supra.)

Therapeutics

[0081] Chemical and structural similarity, in the context of the PDZ domains and aPKC binding region, exists between regions of ARP-1 (1555118; SEQ ID NO:l), ARP-2 (2582063; SEQ ID NO:2), rat ASIP (g3868778; SEQ ID NO:62), and human ASIP (g8037915; SEQ ID NO:63) as shown in FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I and 3J. In addition, differential expression of ARP is associated with the digestive system, female reproductive tissue, hemic and immune system, nervous system, respiratory system, and urinary tract and with cancer, particularly bladder transitional cell carcinoma as shown in Tables 1 and 2. ARP clearly plays a role in cancer, particularly bladder transitional cell carcinoma.

[0082] In the treatment of conditions associated with increased expression of ARP it is desirable to decrease expression or protein activity. In one embodiment, the an inhibitor, antagonist or antibody of the protein may be administered to a subject to treat a condition associated with increased expression or activity. In another embodiment, a pharmaceutical composition comprising an inhibitor, antagonist or antibody in conjunction with a pharmaceutical carrier may be administered to a subject to treat a condition associated with the increased expression or activity of the endogenous protein. In an additional embodiment, a vector expressing the complement of the cDNA or fragments thereof may be administered to a subject to treat the disorder.

[0083] In the treatment of conditions associated with decreased expression of ARP it is desirable to increase expression or protein activity. In one embodiment, the protein, an agonist or enhancer may be administered to a subject to treat a condition associated with decreased expression or activity. In another embodiment, a pharmaceutical composition comprising the protein, an agonist or enhancer in conjunction with a pharmaceutical carrier may be administered to a subject to treat a condition associated with the decreased expression or activity of the endogenous protein. In an additional embodiment, a vector expressing cDNA may be administered to a subject to treat the disorder.

[0084] Any of the cDNAs, complementary molecules, or fragments thereof, proteins or portions thereof, vectors delivering these nucleic acid molecules or expressing the proteins, and their ligands may be administered in combination with other therapeutic agents. Selection of the agents for use in combination therapy may be made by one of ordinary skill in the art according to conventional pharmaceutical principles. A combination of therapeutic agents may act synergistically to affect treatment of a particular disorder at a lower dosage of each agent.

Modification of Gene Expression Using Nucleic Acids

[0085] Gene expression may be modified by designing complementary or antisense molecules (DNA, RNA, or PNA) to the control, 5′, 3′, or other regulatory regions of the gene encoding ARP. Oligonucleotides designed with reference to the transcription initiation site are preferred. Similarly, inhibition can be achieved using triple helix base-pairing which inhibits the binding of polymerases, transcription factors, or regulatory molecules (Gee et al In: Huber and Carr (1994) Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp. 163-177). A complementary molecule may also be designed to block translation by preventing binding between ribosomes and mRNA. In one alternative, a library or plurality of cDNAs or fragments thereof may be screened to identify those which specifically bind a regulatory, nontranslated sequence.

[0086] Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA followed by endonucleolytic cleavage at sites such as GUA, GUU, and GUC. Once such sites are identified, an oligonucleotide with the same sequence may be evaluated for secondary structural features which would render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing their hybridization with complementary oligonucleotides using ribonuclease protection assays.

[0087] Complementary nucleic acids and ribozymes of the invention may be prepared via recombinant expression, in vitro or in vivo, or using solid phase phosphoramidite chemical synthesis. In addition, RNA molecules may be modified to increase intracellular stability and half-life by addition of flanking sequences at the 5′ and/or 3′ ends of the molecule or by the use of phosphorothioate or 2′O methyl rather than phosphodiesterase linkages within the backbone of the molecule. Modification is inherent in the production of PNAs and can be extended to other nucleic acid molecules. Either the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, and or the modification of adenine, cytidine, guanine, thymine, and uridine with acetyl-, methyl-, thio- groups renders the molecule less available to endogenous endonucleases.

Screening and Purification Assays

[0088] The cDNA encoding ARP may be used to screen a library of molecules or compounds for specific binding affinity. The libraries may be aptamers, DNA molecules, RNA molecules, PNAs, peptides, proteins such as transcription factors, enhancers, repressors, and other ligands which regulate the activity, replication, transcription, or translation of the cDNA in the biological system. The assay involves combining the cDNA or a fragment thereof with the library of molecules under conditions allowing specific binding, and detecting specific binding to identify at least one molecule which specifically binds the single stranded or, if appropriate, double stranded molecule.

[0089] In one embodiment, the cDNA of the invention may be incubated with a plurality of purified molecules or compounds and binding activity determined by methods well known in the art, e.g., a gel-retardation assay (U.S. Pat. No. 6,010,849) or a reticulocyte lysate transcriptional assay. In another embodiment, the cDNA may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the cDNA and a molecule or compound in the nuclear extract is initially determined by gel shift assay and may be later confirmed by recovering and raising antibodies against that molecule or compound. When these antibodies are added into the assay, they cause a supershift in the gel-retardation assay.

[0090] In another embodiment, the cDNA may be used to purify a molecule or compound using affinity chromatography methods well known in the art. In one embodiment, the cDNA is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the cDNA. The molecule or compound which is bound to the cDNA may be released from the cDNA by increasing the salt concentration of the flow-through medium and collected.

[0091] In a further embodiment, the protein or a portion thereof may be used to purify a ligand from a sample. A method for using a protein or a portion thereof to purify a ligand would involve combining the protein or a portion thereof with a sample under conditions to allow specific binding, detecting specific binding between the protein and ligand, recovering the bound protein, and using an appropriate chaotropic agent to separate the protein from the purified ligand.

[0092] In a preferred embodiment, ARP or a portion thereof may be used to screen a plurality of molecules or compounds in any of a variety of screening assays. The portion of the protein employed in such screening may be free in solution, affixed to an abiotic or biotic substrate (e.g. borne on a cell surface), or located intracellularly. For example, in one method, viable or fixed prokaryotic host cells that are stably transformed with recombinant nucleic acids that have expressed and positioned a peptide on their cell surface can be used in screening assays. The cells are screened against a plurality or libraries of ligands and the specificity of binding or formation of complexes between the expressed protein and the ligand may be measured. Specific binding between the protein and molecule may be measured. Depending on the kind of library being screened, the assay may be used to identify DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs or any other ligand, which specifically binds the protein.

[0093] In one aspect, this invention comtemplates a method for high throughput screening using very small assay volumes and very small amounts of test compound as described in U.S. Pat. No. 5,876,946, incorporated herein by reference. This method is used to screen large numbers of molecules and compounds via specific binding. In another aspect, this invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein or oligopeptide or portion thereof. Molecules or compounds identified by screening may be used in a mammalian model system to evaluate their toxicity, diagnostic, or therapeutic potential.

Pharmacology

[0094] Pharmaceutical compositions are those substances wherein the active ingredients are contained in an effective amount to achieve a desired and intended purpose. The determination of an effective dose is well within the capability of those skilled in the art. For any compound, the therapeutically effective dose may be estimated initially either in cell culture assays or in animal models. The animal model is also used to achieve a desirable concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans.

[0095] A therapeutically effective dose refers to that amount of protein or inhibitor which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity of such agents may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it may be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceutical compositions which exhibit large therapeutic indexes are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.

Model Systems

[0096] Animal models may be used as bioassays where they exhibit a phenotypic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models, and most infectious agent, cancer, drug, and toxicity studies are performed on rodents such as rats or mice because of low cost, availability, lifespan, reproductive potential, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of under- or over-expression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to over-express a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene.

Toxicology

[0097] Toxicology is the study of the effects of agents on living systems. The majority of toxicity studies are performed on rats or mice. Observation of qualitative and quantitative changes in physiology, behavior, homeostatic processes, and lethality in the rats or mice are used to generate a toxicity profile and to assess potential consequences on human health following exposure to the agent.

[0098] Genetic toxicology identifies and analyzes the effect of an agent on the rate of endogenous, spontaneous, and induced genetic mutations. Genotoxic agents usually have common chemical or physical properties that facilitate interaction with nucleic acids and are most harmful when chromosomal aberrations are transmitted to progeny. Toxicological studies may identify agents that increase the frequency of structural or functional abnormalities in the tissues of the progeny if administered to either parent before conception, to the mother during pregnancy, or to the developing organism. Mice and rats are most frequently used in these tests because their short reproductive cycle allows the production of the numbers of organisms needed to satisfy statistical requirements.

[0099] Acute toxicity tests are based on a single administration of an agent to the subject to determine the symptomology or lethality of the agent. Three experiments are conducted: 1) an initial dose-range-finding experiment, 2) an experiment to narrow the range of effective doses, and 3) a final experiment for establishing the dose-response curve.

[0100] Subchronic toxicity tests are based on the repeated administration of an agent. Rat and dog are commonly used in these studies to provide data from species in different families. With the exception of carcinogenesis, there is considerable evidence that daily administration of an agent at high-dose concentrations for periods of three to four months will reveal most forms of toxicity in adult animals.

[0101] Chronic toxicity tests, with a duration of a year or more, are used to demonstrate either the absence of toxicity or the carcinogenic potential of an agent. When studies are conducted on rats, a minimum of three test groups plus one control group are used, and animals are examined and monitored at the outset and at intervals throughout the experiment.

Transgenic Animal Models

[0102] Transgenic rodents that over-express or under-express a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents. (See, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No. 5,767,337.) In some cases, the introduced gene may be activated at a specific time in a specific tissue type during fetal or postnatal development. Expression of the transgene is monitored by analysis of phenotype, of tissue-specific mRNA expression, or of serum and tissue protein levels in transgenic animals before, during, and after challenge with experimental drug therapies.

Embryonic Stem Cells

[0103] Embryonic (ES) stem cells isolated from rodent embryos retain the potential to form embryonic tissues. When ES cells are placed inside a carrier embryo, they resume normal development and contribute to tissues of the live-born animal. ES cells are the preferred cells used in the creation of experimental knockout and knockin rodent strains. Mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and are grown under culture conditions well known in the art. Vectors used to produce a transgenic strain contain a disease gene candidate and a marker gen, the latter serves to identify the presence of the introduced disease gene. The vector is transformed into ES cells by methods well known in the art, and transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.

[0104] ES cells derived from human blastocysts may be manipulated in vitro to differentiate into at least eight separate cell lineages. These lineages are used to study the differentiation of various cell types and tissues in vitro, and they include endoderm, mesoderm, and ectodermal cell types which differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes.

Knockout Analysis

[0105] In gene knockout analysis, a region of a mammalian gene is enzymatically modified to include a non-mammalian gene such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288-1292). The modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination. The inserted sequence disrupts transcription and translation of the endogenous gene. Transformed cells are injected into rodent blastulae, and the blastulae are implanted into pseudopregnant dams. Transgenic progeny are crossbred to obtain homozygous inbred lines which lack a functional copy of the mammalian gene. In one example, the mammalian gene is a human gene.

Knockin Analysis

[0106] ES cells can be used to create knockin humanized animals (pigs) or transgenic animal models (mice or rats) of human diseases. With knockin technology, a region of a human gene is injected into animal ES cells, and the human sequence integrates into the animal cell genome. Transformed cells are injected into blastulae and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of the analogous human condition. These methods have been used to model several human diseases.

Non-Human Primate Model

[0107] The field of animal testing deals with data and methodology from basic sciences such as physiology, genetics, chemistry, pharmacology and statistics. These data are paramount in evaluating the effects of therapeutic agents on non-human primates as they can be related to human health. Monkeys are used as human surrogates in vaccine and drug evaluations, and their responses are relevant to human exposures under similar conditions. Cynomolgus and Rhesus monkeys (Macaca fascicularis and Macaca mulatta, respectively) and Common Marmosets (Callithrix iacchus) are the most common non-human primates (NHPs) used in these investigations. Since great cost is associated with developing and maintaining a colony of NHPs, early research and toxicological studies are usually carried out in rodent models. In studies using behavioral measures such as drug addiction, NHPs are the first choice test animal. In addition, NHPs and individual humans exhibit differential sensitivities to many drugs and toxins and can be classified as a range of phenotypes from “extensive metabolizers” to “poor metabolizers” of these agents.

[0108] In additional embodiments, the cDNAs which encode the protein may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of cDNAs that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.

EXAMPLES

[0109] The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention. For purposes of example, preparation of the human bladder tumor (BLADTUT04) and normalized breast (BRSTNON02) libraries will be described.

[0110] I cDNA Library Construction

Bladder Tumor Library

[0111] The BLADTUT04 cDNA library was constructed from bladder tumor tissue obtained from a 60 year-old Caucasian male who had a transitional cell carcinoma in the left bladder wall. The frozen tissue was homogenized and lysed using a POLYTRON homogenizer (Brinkmann Instruments, Westbury N.J.). The reagents and extraction procedures were used as supplied in the RNA Isolation kit (Stratagene). The lysate was centrifuged over a 5.7 M CsCl cushion using an SW28 rotor in an L8-70M ultracentrifuge (Beckman Coulter, Fullerton Calif.) for 18 hr at 25,000 rpm at ambient temperature. The RNA was extracted twice with phenol chloroform, pH 8.0, and once with acid phenol, pH 4.7; precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol; resuspended in water; and treated with DNase for 15 min at 37C. The RNA was isolated with the OLIGOTEX kit (Qiagen, Chatsworth Calif.) and used to construct the cDNA library.

[0112] The mRNA was handled according to the recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies) which contains a NotI primer-adaptor designed to prime the first strand cDNA synthesis at the poly(A) tail of mRNAs. Double stranded cDNA was blunted, ligated to EcoRI adaptors and digested with NotI (New England Biolabs, Beverly Mass.). The cDNAs were fractionated on a SEPHAROSE CL4B column (APB), and those cDNAs exceeding 400 bp were ligated into pINCY plasmid (Incyte Genomics). The plasmid pINCY was subsequently transformed into DH5α competent cells (Life Technologies).

Normalized Breast

[0113] About 1.2×10⁶ independent clones of the pooled BRSTNOT34 and BRSTNOT35 plasmid libraries in E. coli strain DH12S competent cells (Life Technologies) were grown in liquid culture under carbenicillin (25 mg/l) and methicillin (1 mg/ml) selection following transformation by electroporation. To reduce the number of excess cDNA copies according to their abundance levels in the library, the cDNA library was normalized in two rounds according to the procedure of Soares et al.(1994; Proc Natl Acad Sci 91:9228-9232) and Bonaldo et al.(1996; Genome Research 6:791-806), with the following modifications. The primer to template ratio in the primer extension reaction was increased from 2:1 to 300:1. The reannealing hybridization was extended from 13 to 48 hr. The single stranded DNA circles of the normalized library were purified by hydroxyapatite chromatography and converted to partially double-stranded by random priming, ligated into pINCY plasmid and electroporated into DH12S competent cells (Life Technologies).

[0114] II Construction of pINCY Plasmid

[0115] The plasmid was constructed by digesting the pSPORT1 plasmid (Life Technologies) with EcoRI restriction enzyme (New England Biolabs, Beverly Mass.) and filling the overhanging ends using Klenow enzyme (New England Biolabs) and 2′-deoxynucleotide 5′-triphosphates (dNTPs). The plasmid was self-ligated and transformed into the bacterial host, E. coli strain JM109.

[0116] An intermediate plasmid produced by the bacteria (pSPORT 1-ΔRI) showed no digestion with EcoRI and was digested with Hind III (New England Biolabs) and the overhanging ends were again filled in with Klenow and dNTPs. A linker sequence was phosphorylated, ligated onto the 5′ blunt end, digested with EcoRI, and self-ligated. Following transformation into JM109 host cells, plasmids were isolated and tested for preferential digestibility with EcoRI, but not with Hind III. A single colony that met this criteria was designated pINCY plasmid.

[0117] After testing the plasmid for its ability to incorporate cDNAs from a library prepared using NotI and EcoRI restriction enzymes, several clones were sequenced; and a single clone containing an insert of approximately 0.8 kb was selected from which to prepare a large quantity of the plasmid. After digestion with NotI and EcoRI, the plasmid was isolated on an agarose gel and purified using a QIAQUICK column (Qiagen) for use in library construction.

[0118] III Isolation and Sequencing of cDNA Clones

[0119] Plasmid DNA was released from the cells and purified using either the MINIPREP kit (Edge Biosystems, Gaithersburg Md.) or the REAL PREP 96 plasmid kit (Qiagen). This kit consists of a 96-well block with reagents for 960 purifications. The recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile TERRIFIC BROTH (BD Biosciences, Sparks Md.) with carbenicillin at 25 mg/l and glycerol at 0.4%; 2) after inoculation, the cells were cultured for 19 hours and then lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol, samples were transferred to a 96-well block for storage at 4C.

[0120] The cDNAs were prepared for sequencing using the MICROLAB 2200 system (Hamilton) in combination with the DNA ENGINE thermal cyclers (MJ Research). The cDNAs were sequenced by the method of Sanger and Coulson (1975; J Mol Biol 94:441-448) using an ABI PRISM 377 sequencing system (Applied Biosystems) or the MEGABACE 1000 DNA sequencing system (APB). Most of the isolates were sequenced according to standard ABI protocols and kits (Applied Biosystems) with solution volumes of 0.25×−1.0×concentrations. In the alternative, cDNAs were sequenced using solutions and dyes from APB.

[0121] IV Extension of cDNA Sequences

[0122] The cDNAs were extended using the cDNA clone and oligonucleotide primers. One primer was synthesized to initiate 5′ extension of the known fragment, and the other, to initiate 3′ extension of the known fragment. The initial primers were designed using OLIGO primer analysis software (Molecular Biology Insights), to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68C. to about 72C. Any stretch of nucleotides that would result in hairpin structures and primer-primer dimerizations was avoided.

[0123] Selected cDNA libraries were used as templates to extend the sequence. If more than one extension was necessary, additional or nested sets of primers were designed. Preferred libraries have been size-selected to include larger cDNAs and random primed to contain more sequences with 5′ or upstream regions of genes. Genomic libraries are used to obtain regulatory elements, especially extension into the 5′ promoter binding region.

[0124] High fidelity amplification was obtained by PCR using methods such as that taught in U.S. Pat. No. 5,932,451. PCR was performed in 96-well plates using the DNA ENGINE thermal cycler (MJ Research). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg²⁺, (NH₄)₂SO₄, and β-mercaptoethanol, Taq DNA polymerase (APB), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B (Incyte Genomics): Step 1: 94C., three min; Step 2: 94C., 15 sec; Step 3: 60C., one min; Step 4: 68C, two min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68C, five min; Step 7: storage at 4C. In the alternative, the parameters for primer pair T7 and SK+(Stratagene) were as follows: Step 1: 94C., three min; Step 2: 94C., 15 sec; Step 3: 57C, one min; Step 4: 68C., two min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68C, five min; Step 7: storage at 4C.

[0125] The concentration of DNA in each well was determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% reagent in 1×TE, v/v; Molecular Probes) and 0.5 μl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning, Acton Mass.) and allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II (Labsystems Oy) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 μl to 10 μl aliquot of the reaction mixture was analyzed by electrophoresis on a 1% agarose mini-gel to determine which reactions were successful in extending the sequence.

[0126] The extended clones were desalted, concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wis.), and sonicated or sheared prior to religation into pUC1 8 vector (APB). For shotgun sequences, the digested nucleotide sequences were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and the agar was digested with AGARACE enzyme (Promega). Extended clones were religated using T4 DNA ligase (New England Biolabs) into pUC18 vector (APB), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into E. coli competent cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37C. in 384-well plates in LB/2×carbenicillin liquid media.

[0127] The cells were lysed, and DNA was amplified using primers, Taq DNA polymerase (APB) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94C., three min; Step 2: 94C., 15 sec; Step 3: 60C., one min; Step 4: 72C., two min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72C., five min; Step 7: storage at 4C. DNA was quantified using PICOGREEN quantitative reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the conditions described above. Samples were diluted with 20% dimethylsulfoxide (DMSO; 1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT cycle sequencing kit (APB) or the ABI PRISM BIGDYE terminator cycle sequencing kit (Applied Biosystems).

[0128] V Homology Searching of cDNA Clones and Their Deduced Proteins

[0129] The cDNAs of the Sequence Listing or their deduced amino acid sequences were used to query databases such as GenBank, SwissProt, BLOCKS, and the like. These databases that contain previously identified and annotated sequences or domains were searched using BLAST or BLAST 2 (Altschul et al. supra; Altschul, supra) to produce alignments and to determine which sequences were exact matches or homologs. The alignments were to sequences of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. Alternatively, algorithms such as the one described in Smith and Smith (1992, Protein Engineering 5:35-51) could have been used to deal with primary sequence patterns and secondary structure gap penalties. All of the sequences disclosed in this application have lengths of at least 49 nucleotides, and no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T).

[0130] As detailed in Karlin (supra), BLAST matches between a query sequence and a database sequence were evaluated statistically and only reported when they satisfied the threshold of 10⁻²⁵ for nucleotides and 10⁻¹⁴ for peptides. Homology was also evaluated by product score calculated as follows: the % nucleotide or amino acid identity [between the query and reference sequences] in BLAST is multiplied by the % maximum possible BLAST score [based on the lengths of query and reference sequences] and then divided by 100. In comparison with hybridization procedures used in the laboratory, the electronic stringency for an exact match was set at 70, and the conservative lower limit for an exact match was set at approximately 40 (with 1-2% error due to uncalled bases).

[0131] The BLAST software suite, freely available sequence comparison algorithms (NCBI, Bethesda Md.; http://www.ncbi.nlm.nih.gov/gorf/b12.html), includes various sequence analysis programs including “blastn” that is used to align nucleic acid molecules and BLAST 2 that is used for direct pairwise comparison of either nucleic or amino acid molecules. BLAST programs are commonly used with gap and other parameters set to default settings, e.g.: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: -2; Open Gap: 5 and Extension Gap: 2 penalties; Gap x drop-off: 50; Expect: 10; Word Size: 11; and Filter: on. Identity is measured over the entire length of a sequence or some smaller portion thereof. Brenner et al. (1998; Proc Natl Acad Sci 95:6073-6078, incorporated herein by reference) analyzed the BLAST for its ability to identify structural homologs by sequence identity and found 30% identity is a reliable threshold for sequence alignments of at least 150 residues and 40%, for alignments of at least 70 residues.

[0132] Putative ASIP-related proteins were initially identified by running the Genscan gene identification program against public genomic sequence databases (e.g., gbpri and gbhtg). Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (See Burge and Karlin (1997) J Mol Biol 268:78-94, and Burge and Karlin (1998) Curr Opin Struct Biol 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode ASIP-related proteins, the encoded polypeptides were analyzed by querying against PFAM models for ASIP-related proteins. Potential ASIP-related proteins were also identified by homology to Incyte cDNA sequences that had been annotated as ASIP-related proteins. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence. Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example III. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.

[0133] The cDNAs of this application were compared with assembled consensus sequences or templates found in the LIFESEQ GOLD database. Component sequences from cDNA, extension, full length, and shotgun sequencing projects were subjected to PHRED analysis and assigned a quality score. All sequences with an acceptable quality score were subjected to various pre-processing and editing pathways to remove low quality 3′ ends, vector and linker sequences, polyA tails, Alu repeats, mitochondrial and ribosomal sequences, and bacterial contamination sequences. Edited sequences had to be at least 50 bp in length, and low-information sequences and repetitive elements such as dinucleotide repeats, Alu repeats, and the like, were replaced by “Ns” or masked.

[0134] Edited sequences were subjected to assembly procedures in which the sequences were assigned to gene bins. Each sequence could only belong to one bin, and sequences in each bin were assembled to produce a template. Newly sequenced components were added to existing bins using BLAST and CROSSMATCH. To be added to a bin, the component sequences had to have a BLAST quality score greater than or equal to 150 and an alignment of at least 82% local identity. The sequences in each bin were assembled using PHRAP. Bins with several overlapping component sequences were assembled using DEEP PHRAP. The orientation of each template was determined based on the number and orientation of its component sequences.

[0135] Bins were compared to one another and those having local similarity of at least 82% were combined and reassembled. Bins having templates with less than 95% local identity were split. Templates were subjected to analysis by STITCHER/EXON MAPPER algorithms that analyze the probabilities of the presence of splice variants, alternatively spliced exons, splice junctions, differential expression of alternative spliced genes across tissue types or disease states, and the like. Assembly procedures were repeated periodically, and templates were annotated using BLAST against GenBank databases such as GBpri. An exact match was defined as having from 95% local identity over 200 base pairs through 100% local identity over 100 base pairs and a homolog match as having an E-value (or probability score) of ≦1×10⁻⁸. The templates were also subjected to frameshift FASTx against GENPEPT, and homolog match was defined as having an E-value of ≦1×10⁻⁸. Template analysis and assembly was described in U.S. Ser. No. 09/276,534, filed Mar. 25, 1999.

[0136] Following assembly, templates were subjected to BLAST, motif, and other functional analyses and categorized in protein hierarchies using methods described in U.S. Ser. No. 08/812,290 and U.S. Ser. No. 08/811,758, both filed Mar. 6, 1997; in U.S. Ser. No. 08/947,845, filed Oct. 9, 1997; and in U.S. Ser. No. 09/034,807, filed Mar. 4, 1998. Then templates were analyzed by translating each template in all three forward reading frames and searching each translation against the PFAM database of hidden Markov model-based protein families and domains using the HMMER software package (Washington University School of Medicine, St. Louis Mo.; http://pfam.wustl.edu/). The cDNA was further analyzed using MACDNASIS PRO software (Hitachi Software Engineering), and LASERGENE software (DNASTAR) and queried against public databases such as the GenBank rodent, mammalian, vertebrate, prokaryote, and eukaryote databases, SwissProt, BLOCKS, PRINTS, PFAM, and Prosite.

[0137] VI Chromosome Mapping

[0138] Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Généthon are used to determine if any of the cDNAs presented in the Sequence Listing have been mapped. Any of the fragments of the cDNA encoding ARP that have been mapped result in the assignment of all related regulatory and coding sequences mapping to the same location. The genetic map locations are described as ranges, or intervals, of human chromosomes. The map position of an interval, in cM (which is roughly equivalent to 1 megabase of human DNA), is measured relative to the terminus of the chromosomal p-arm.

[0139] VII Hybridization Technologies and Analyses

Immobilization of cDNAs on a Substrate

[0140] The cDNAs are applied to a substrate by one of the following methods. A mixture of cDNAs is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary transfer. Alternatively, the cDNAs are individually ligated to a vector and inserted into bacterial host cells to form a library. The cDNAs are then arranged on a substrate by one of the following methods. In the first method, bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane. The membrane is placed on LB agar containing selective agent (carbenicillin, kanamycin, ampicillin, or chloramphenicol depending on the vector used) and incubated at 37C. for 16 hr. The membrane is removed from the agar and consecutively placed colony side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH), neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2×SSC for 10 min each. The membrane is then UV irradiated in a STRATALINKER UV-crosslinker (Stratagene).

[0141] In the second method, cDNAs are amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert. PCR amplification increases a starting concentration of 1-2 ng nucleic acid to a final quantity greater than 5 μg. Amplified nucleic acids from about 400 bp to about 5000 bp in length are purified using SEPHACRYL-400 beads (APB). Purified nucleic acids are arranged on a nylon membrane manually or using a dot/slot blotting manifold and suction device and are immobilized by denaturation, neutralization, and UV irradiation as described above. Purified nucleic acids are robotically arranged and immobilized on polymer-coated glass slides using the procedure described in U.S. Pat. No. 5,807,522. Polymer-coated slides are prepared by cleaning glass microscope slides (Corning, Acton Mass.) by ultrasound in 0.1% SDS and acetone, etching in 4% hydrofluoric acid (VWR Scientific Products, West Chester Pa.), coating with 0.05% aminopropyl silane (Sigma Aldrich) in 95% ethanol, and curing in a 110C. oven. The slides are washed extensively with distilled water between and after treatments. The nucleic acids are arranged on the slide and then immobilized by exposing the array to UV irradiation using a STRATALINKER UV-crosslinker (Stratagene). Arrays are then washed at room temperature in 0.2% SDS and rinsed three times in distilled water. Non-specific binding sites are blocked by incubation of arrays in 0.2% casein in phosphate buffered saline (PBS; Tropix, Bedford Mass.) for 30 min at 60C.; then the arrays are washed in 0.2% SDS and rinsed in distilled water as before.

Probe Preparation for Membrane Hybridization

[0142] Hybridization probes derived from the cDNAs of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the cDNAs to a concentration of 40-50 ng in 45 μl TE buffer, denaturing by heating to 100C. for five min, and briefly centrifuging. The denatured cDNA is then added to a REDIPRIME tube (APB), gently mixed until blue color is evenly distributed, and briefly centrifuged. Five μl of [³²P]dCTP is added to the tube, and the contents are incubated at 37C. for 10 min. The labeling reaction is stopped by adding 5 μl of 0.2M EDTA, and probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB). The purified probe is heated to 100C. for five min, snap cooled for two min on ice, and used in membrane-based hybridizations as described below.

Probe Preparation for Polymer Coated Slide Hybridization

[0143] Hybridization probes derived from mRNA isolated from samples are employed for screening cDNAs of the Sequence Listing in array-based hybridizations. Probe is prepared using the GEMbright kit (Incyte Genomics) by diluting mRNA to a concentration of 200 ng in 9 μl TE buffer and adding 5 μl 5×buffer, 1 μl 0.1 M DTT, 3 μl Cy3 or Cy5 labeling mix, 1μl RNase inhibitor, 1 μl reverse transcript and 5 μl 1 ×yeast control mRNAs. Yeast control mRNAs are synthesized by in vitro transcription from noncoding yeast genomic DNA (W. Lei, unpublished). As quantitative controls, one set of control mRNAs at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng are diluted into reverse transcription reaction mixture at ratios of 1:100,000, 1:10,000, 1:1000, and 1:100 (w/w) to sample mRNA respectively. To examine mRNA differential expression patterns, a second set of control mRNAs are diluted into reverse transcription reaction mixture at ratios of 1:3,3:1, 1:10, 10:1, 1:25, and 25:1 (w/w). The reaction mixture is mixed and incubated at 37C. for two hr. The reaction mixture is then incubated for 20 min at 85C., and probes are purified using two successive CHROMA SPIN+TE 30 columns (Clontech, Palo Alto Calif.). Purified probe is ethanol precipitated by diluting probe to 90 μl in DEPC-treated water, adding 2 μl 1 mg/ml glycogen, 60 μl 5 M sodium acetate, and 300 μl 100% ethanol. The probe is centrifuged for 20 min at 20,800×g, and the pellet is resuspended in 12 μl resuspension buffer, heated to 65C. for five min, and mixed thoroughly. The probe is heated and mixed as before and then stored on ice. Probe is used in high density array-based hybridizations as described below.

Membrane-based Hybridization

[0144] Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and 1×high phosphate buffer (0.5 M NaCl, 0.1 M Na₂HPO₄, 5 mM EDTA, pH 7) at 55C. for two hr. The probe, diluted in 15 ml fresh hybridization solution, is then added to the membrane. The membrane is hybridized with the probe at 55C. for 16 hr. Following hybridization, the membrane is washed for 15 min at 25C. in 1 mM Tris (pH 8.0), 1 % Sarkosyl, and four times for 15 min each at 25C. in 1 mM Tris (pH 8.0). To detect hybridization complexes, XOMAT-AR film (Eastman Kodak, Rochester N.Y.) is exposed to the membrane overnight at −70C., developed, and examined visually.

Polymer Coated Slide-based Hybridization

[0145] Probe is heated to 65C. for five min, centrifuged five min at 9400 rpm in a 5415C. microcentrifuge (Eppendorf Scientific, Westbury N.Y.), and then 18 μl is aliquoted onto the array surface and covered with a coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 μl of 5×SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hr at 60C. The arrays are washed for 10 min at 45C. in 1×SSC, 0.1 % SDS, and three times for 10 min each at 45C. in 0.1×SSC, and dried.

[0146] Hybridization reactions are performed in absolute or differential hybridization formats. In the absolute hybridization format, probe from one sample is hybridized to array elements, and signals are detected after hybridization complexes form. Signal strength correlates with probe mRNA levels in the sample. In the differential hybridization format, differential expression of a set of genes in two biological samples is analyzed. Probes from the two samples are prepared and labeled with different labeling moieties. A mixture of the two labeled probes is hybridized to the array elements, and signals are examined under conditions in which the emissions from the two different labels are individually detectable. Elements on the array that are hybridized to substantially equal numbers of probes derived from both biological samples give a distinct combined fluorescence (Shalon WO95/35505).

[0147] Hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara Calif.) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser light is focused on the array using a 20×microscope objective (Nikon, Melville N.Y.). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective with a resolution of 20 micrometers. In the differential hybridization format, the two fluorophores are sequentially excited by the laser. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. The sensitivity of the scans is calibrated using the signal intensity generated by the yeast control mRNAs added to the probe mix. A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000.

[0148] The output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Norwood Mass.) installed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using the emission spectrum for each fluorophore. A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS program (Incyte Genomics).

[0149] VIII Electronic Analysis

[0150] BLAST was used to search for identical or related molecules in the GenBank or LIFESEQ databases (Incyte Genomics). The product score for human and rat sequences was calculated as follows: the BLAST score is multiplied by the % nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences), such that a 100% alignment over the length of the shorter sequence gives a product score of 100. The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and with a product score of at least 70, the match will be exact. Similar or related molecules are usually identified by selecting those which show product scores between 8 and 40.

[0151] Electronic northern analysis was performed at a product score of 70 as shown in Tables 1 and 2. All sequences and cDNA libraries in the LIFESEQ database were categorized by system, organ/tissue and cell type. The categories included cardiovascular system, connective tissue, digestive system, embryonic structures, endocrine system, exocrine glands, female and male genitalia, germ cells, hemic/immune system, liver, musculoskeletal system, nervous system, pancreas, respiratory system, sense organs, skin, stomatognathic system, unclassified/mixed, and the urinary tract. For each category, the number of libraries in which the sequence was expressed were counted and shown over the total number of libraries in that category. In a non-normalized library, expression levels of two or more are significant.

IX Complementary Molecules

[0152] Molecules complementary to the cDNA, from about 5 (PNA) to about 5000 bp (complement of a cDNA insert), are used to detect or inhibit gene expression. These molecules are selected using OLIGO primer analysis software (Molecular Biology Insights). Detection is described in Example VII. To inhibit transcription by preventing promoter binding, the complementary molecule is designed to bind to the most unique 5′ sequence and includes nucleotides of the 5′ UTR upstream of the initiation codon of the open reading frame. Complementary molecules include genomic sequences (such as enhancers or introns) and are used in “triple helix” base pairing to compromise the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. To inhibit translation, a complementary molecule is designed to prevent ribosomal binding to the mRNA encoding the protein.

[0153] Complementary molecules are placed in expression vectors and used to transform a cell line to test efficacy; into an organ, tumor, synovial cavity, or the vascular system for transient or short term therapy; or into a stem cell, zygote, or other reproducing lineage for long term or stable gene therapy. Transient expression lasts for a month or more with a non-replicating vector and for three months or more if appropriate elements for inducing vector replication are used in the transformation/expression system.

[0154] Stable transformation of appropriate dividing cells with a vector encoding the complementary molecule produces a transgenic cell line, tissue, or organism (U.S. Pat. No. 4,736,866). Those cells that assimilate and replicate sufficient quantities of the vector to allow stable integration also produce enough complementary molecules to compromise or entirely eliminate activity of the cDNA encoding the protein.

[0155] X Selection of Sequences, Microarray Preparation and Use

[0156] Incyte clones represent template sequences derived from the LIFESEQ GOLD assembled human sequence database (Incyte Genomics). In cases where more than one clone was available for a particular template, the 5′-most clone in the template was used on the microarray. The HUMAN GENOME GEM series 1-3 microarrays (Incyte Genomics) contain 28,626 array elements which represent 10,068 annotated clusters and 18,558 unannotated clusters. For the UNIGEM series microarrays (Incyte Genomics), Incyte clones were mapped to non-redundant Unigene clusters (Unigene database (build 46), NCBI; Shuler (1997) J Mol Med 75:694-698), and the 5′ clone with the strongest BLAST alignment (at least 90% identity and 100 bp overlap) was chosen, verified, and used in the construction of the microarray. The UNIGEM V microarray (Incyte Genomics) contains 7075 array elements which represent 4610 annotated genes and 2,184 unannotated clusters.

[0157] To construct microarrays, cDNAs were amplified from bacterial cells using primers complementary to vector sequences flanking the cDNA insert. Thirty cycles of PCR increased the initial quantity of cDNAs from 1-2 ng to a final quantity of greater than 5 μg. Amplified cDNAs were then purified using SEPHACRYL-400 columns (APB ). Purified cDNAs were immobilized on polymer-coated glass slides. Glass microscope slides (Corning, Corning N.Y.) were cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides were etched in 4% hydrofluoric acid (VWR Scientific Products, West Chester Pa.), washed thoroughly in distilled water, and coated with 0.05% aminopropyl silane (Sigma Aldrich) in 95% ethanol. Coated slides were cured in a 110° C. oven. cDNAs were applied to the coated glass substrate using a procedure described in U.S. Pat. No. 5,807,522. One microliter of the cDNA at an average concentration of 100 ng/μl was loaded into the open capillary printing element by a high-speed robotic apparatus which then deposited about 5 nl of cDNA per slide.

[0158] Microarrays were UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene), and then washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites were blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (Tropix, Bedford Mass.) for 30 minutes at 60° C. followed by washes in 0.2% SDS and distilled water as before.

[0159] XI Preparation of Samples

Human BT20 Cells

[0160] BT-20 is a breast carcinoma cell line derived in vitro from the cells emigrating out of thin slices of a tumor mass isolated from a 74 year old female. BT20 cells were treated with EGF at a concentration of 50 ng/ml for 4, 8, 12, 24, 36 and 48 hours. In all cases mRNA from untreated BT20 cells were prepared in parallel.

[0161] XII Expression of ARP

[0162] Expression and purification of the protein are achieved using either a mammalian cell expression system or an insect cell expression system. The pUB6/V5-His vector system (Invitrogen, Carlsbad Calif.) is used to express ARP in CHO cells. The vector contains the selectable bsd gene, multiple cloning sites, the promoter/enhancer sequence from the human ubiquitin C gene, a C-terminal V5 epitope for antibody detection with anti-V5 antibodies, and a C-terminal polyhistidine (6×His) sequence for rapid purification on PROBOND resin (Invitrogen). Transformed cells are selected on media containing blasticidin.

[0163]Spodoptera frugiperda (Sf9) insect cells are infected with recombinant Autographica californica nuclear polyhedrosis virus (baculovirus). The polyhedrin gene is replaced with the cDNA by homologous recombination and the polyhedrin promoter drives cDNA transcription. The protein is synthesized as a fusion protein with 6×his which enables purification as described above. Purified protein is used in the following activity and to make antibodies

[0164] XIII Production of Antibodies

[0165] ARP is purified using polyacrylamide gel electrophoresis and used to immunize mice or rabbits. Antibodies are produced using the protocols below. Alternatively, the amino acid sequence of ARP is analyzed using LASERGENE software (DNASTAR) to determine regions of high antigenicity. An antigenic epitope, usually found near the C-terminus or in a hydrophilic region is selected, synthesized, and used to raise antibodies. Typically, epitopes of about 15 residues in length are produced using an ABI 431A peptide synthesizer (Applied Biosystems) using Fmoc-chemistry and coupled to KLH (Sigma-Aldrich) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester to increase antigenicity.

[0166] Rabbits are immunized with the epitope-KLH complex in complete Freund's adjuvant. Immunizations are repeated at intervals thereafter in incomplete Freund's adjuvant. After a minimum of seven weeks for mouse or twelve weeks for rabbit, antisera are drawn and tested for antipeptide activity. Testing involves binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG. Methods well known in the art are used to determine antibody titer and the amount of complex formation.

[0167] XIV Purification of Naturally Occurring Protein Using Specific Antibodies

[0168] Naturally occurring or recombinant protein is purified by immunoaffinity chromatography using antibodies which specifically bind the protein. An immunoaffinity column is constructed by covalently coupling the antibody to CNBr-activated SEPHAROSE resin (APB). Media containing the protein is passed over the immunoaffinity column, and the column is washed using high ionic strength buffers in the presence of detergent to allow preferential absorbance of the protein. After coupling, the protein is eluted from the column using a buffer of pH 2-3 or a high concentration of urea or thiocyanate ion to disrupt antibody/protein binding, and the protein is collected.

[0169] XV Screening Molecules for Specific Binding with the cDNA or Protein

[0170] The cDNA, or fragments thereof, or the protein, or portions thereof, are labeled with ³²P-dCTP, Cy3-dCTP, or Cy5-dCTP (APB), or with BIODIPY or ITC (Molecular Probes, Eugene Oreg.), respectively. Libraries of candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled cDNA or protein. After incubation under conditions for either a nucleic acid or amino acid sequence, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed, and the ligand is identified. Data obtained using different concentrations of the nucleic acid or protein are used to calculate affinity between the labeled nucleic acid or protein and the bound molecule.

[0171] XVI Two-Hybrid Screen

[0172] A yeast two-hybrid system, MATCHMAKER LexA Two-Hybrid system (Clontech Laboratories, Palo Alto Calif.), is used to screen for peptides that bind the protein of the invention. A cDNA encoding the protein is inserted into the multiple cloning site of a pLexA vector, ligated, and transformed into E. coli. cDNA, prepared from mRNA, is inserted into the multiple cloning site of a pB42AD vector, ligated, and transformed into E. coli to construct a cDNA library. The pLexA plasmid and pB42AD-cDNA library constructs are isolated from E. coli and used in a 2:1 ratio to co-transform competent yeast EGY48[p8op-lacZ] cells using a polyethylene glycol/lithium acetate protocol. Transformed yeast cells are plated on synthetic dropout (SD) media lacking histidine (-His), tryptophan (-Trp), and uracil (-Ura), and incubated at 30C. until the colonies have grown up and are counted. The colonies are pooled in a minimal volume of 1×TE (pH 7.5), replated on SD/-1 His/-Leu/-Trp/-Ura media supplemented with 2% galactose (Gal), 1% raffinose (Raf), and 80 mg/ml 5-bromo-4-chloro-3-indolyl β-d -galactopyranoside (X-Gal), and subsequently examined for growth of blue colonies. Interaction between expressed protein and cDNA fusion proteins activates expression of a LEU2 reporter gene in EGY48 and produces colony growth on media lacking leucine (-Leu). Interaction also activates expression of β-galactosidase from the p8op-lacZ reporter construct that produces blue color in colonies grown on X-Gal.

[0173] Positive interactions between expressed protein and cDNA fusion proteins are verified by isolating individual positive colonies and growing them in SD/-Trp/-Ura liquid medium for 1 to 2 days at 30C. A sample of the culture is plated on SD/-Trp/-Ura media and incubated at 30C. until colonies appear. The sample is replica-plated on SD/-Trp/-Ura and SD/-His/-Trp/-Ura plates. Colonies that grow on SD containing histidine but not on media lacking histidine have lost the pLexA plasmid. Histidine-requiring colonies are grown on SD/Gal/Raf/X-Gal/-Trp/-Ura, and white colonies are isolated and propagated. The pB42AD-cDNA plasmid, which contains a cDNA encoding a protein that physically interacts with the protein, is isolated from the yeast cells and characterized.

[0174] XVII ARP Assay

[0175] ARP activity is determined in a ligand-binding assay using candidate ligand molecules such as the aPKCs, PKCζ and PKCλ, in the presence of ¹²⁵I-labeled ARP. ARP is labeled with ¹²⁵I Bolton-Hunter reagent (Bolton and Hunter (1973) Biochem J 133:529-539). Candidate ARP molecules, previously arrayed in the wells of a multi-well plate, are incubated with the labeled ARP, washed, and any wells with labeled ARP complex are assayed. Data obtained using different concentrations of ARP are used to calculate values for the number, affinity, and association of ARP with the candidate molecules.

[0176] All patents and publications mentioned in the specification are incorporated by reference herein. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims. TABLE 1 Clone Abs Pct Tissue Category Count Found in Abund Abund Cardiovascular System 266190 1/68 1 0.0004 Connective Tissue 144645 0/47 0 0.0000 Digestive System 501101  4/148 4 0.0008 Embryonic Structures 106713 0/21 0 0.0000 Endocrine System 225386 0/53 0 0.0000 Exocrine Glands 254635 1/64 1 0.0004 Reproductive, Female 427284  2/106 2 0.0005 Reproductive, Male 448207  1/114 1 0.0002 Germ Cells  38282 1/5  1 0.0026 Hemic and Immune System 680277  2/159 2 0.0003 Liver 109378 0/35 0 0.0000 Musculoskeletal System 159280 0/47 0 0.0000 Nervous System 955753  3/198 5 0.0005 Pancreas 110207 0/24 0 0.0000 Respiratory System 390086 3/93 3 0.0008 Sense Organs  19256 0/8  0 0.0000 Skin  72292 0/15 0 0.0000 Stomatognathic System  12923 0/10 0 0.0000 Unclassified/Mixed 120926 2/13 2 0.0017 Urinary Tract 279062 3/64 5 0.0018 Totals 5321883   23/1292 27  0.0005

[0177] TABLE 2 Clone Abs Pct Library ID Count Library Description Abund Abund BLADTUT04 7884 bladder tumor, TC CA, 3 0.0381 60M, m/BLADNOT05 BLADNOT05 3776 bladder, mw/TC CA, CA in 0 0 situ, 60M, m/BLADTUT04

[0178] TABLE 3 mean log2 DE (Cy5/Cy3) Cy3 Cy5 −0.08 Human, BT20 Line, Untx Human, BT20 Line, t/EGF 4 hr, AdenoCA 50 ng/ml 4 hr, AdenoCA −0.51 Human, BT20 Line, Untx Human, BT20 Line, t/EGF 8 hr, AdenoCA 50 ng/ml 8 hr, AdenoCA −0.61 Human, BT20 Line, Untx Human, BT20 Line, t/EGF 12 hr, AdenoCA 50 ng/ml 12 hr, AdenoCA −0.52 Human, BT20 Line, Untx Human, BT20 Line, t/EGF 24 hr, AdenoCA 50 ng/ml 24 hr, AdenoCA −1.23 Human, BT20 Line, Untx Human, BT20 Line, t/EGF 36 hr, AdenoCA 50 ng/ml 36 hr, AdenoCA −1.04 Human, BT20 Line, Untx Human, BT20 Line, t/EGF 48 hr, AdenoCA 50 ng/ml 48 hr, AdenoCA

[0179]

1 63 1 935 PRT Homo sapiens misc_feature Incyte ID No 1555118CD1 1 Met Lys Val Thr Val Cys Phe Gly Arg Thr Gly Ile Val Val Pro 1 5 10 15 Cys Lys Glu Gly Gln Leu Arg Val Gly Glu Leu Thr Gln Gln Ala 20 25 30 Leu Gln Arg Tyr Leu Lys Thr Arg Glu Lys Gly Pro Gly Tyr Trp 35 40 45 Val Lys Ile His His Leu Glu Tyr Thr Asp Gly Gly Ile Leu Asp 50 55 60 Pro Asp Asp Val Leu Ala Asp Val Val Glu Asp Lys Asp Lys Leu 65 70 75 Ile Ala Val Phe Glu Glu Gln Glu Pro Leu His Lys Ile Glu Ser 80 85 90 Pro Ser Gly Asn Pro Ala Asp Arg Gln Ser Pro Asp Ala Phe Glu 95 100 105 Thr Glu Val Ala Ala Gln Leu Ala Ala Phe Lys Pro Ile Gly Gly 110 115 120 Glu Ile Glu Val Thr Pro Ser Ala Leu Lys Leu Gly Thr Pro Leu 125 130 135 Leu Val Arg Arg Ser Ser Asp Pro Val Pro Gly Pro Pro Ala Asp 140 145 150 Thr Gln Pro Ser Ala Ser His Pro Gly Gly Gln Ser Leu Lys Leu 155 160 165 Val Val Pro Asp Ser Thr Gln Asn Leu Glu Asp Arg Glu Val Leu 170 175 180 Asn Gly Val Gln Thr Glu Leu Leu Thr Ser Pro Arg Thr Lys Asp 185 190 195 Thr Leu Ser Asp Met Thr Arg Thr Val Glu Ile Ser Gly Glu Gly 200 205 210 Gly Pro Leu Gly Ile His Val Val Pro Phe Phe Ser Ser Leu Ser 215 220 225 Gly Arg Ile Leu Gly Leu Phe Ile Arg Gly Ile Glu Asp Asn Ser 230 235 240 Arg Ser Lys Arg Glu Gly Leu Phe His Glu Asn Glu Cys Ile Val 245 250 255 Lys Ile Asn Asn Val Asp Leu Val Asp Lys Thr Phe Ala Gln Ala 260 265 270 Gln Asp Val Phe Arg Gln Ala Met Lys Ser Pro Ser Val Leu Leu 275 280 285 His Val Leu Pro Pro Gln Asn Arg Glu Gln Tyr Glu Lys Ser Val 290 295 300 Ile Gly Ser Leu Asn Ile Phe Gly Asn Asn Asp Gly Val Leu Lys 305 310 315 Thr Lys Val Pro Pro Pro Val His Gly Lys Ser Gly Leu Lys Thr 320 325 330 Ala Asn Leu Thr Gly Thr Asp Ser Pro Glu Thr Asp Ala Ser Ala 335 340 345 Ser Leu Gln Gln Asn Lys Ser Pro Arg Val Pro Arg Leu Gly Gly 350 355 360 Lys Pro Ser Ser Pro Ser Leu Ser Pro Leu Met Gly Phe Gly Ser 365 370 375 Asn Lys Asn Ala Lys Lys Ile Lys Ile Asp Leu Lys Lys Gly Pro 380 385 390 Glu Gly Leu Gly Phe Thr Val Val Thr Arg Asp Ser Ser Ile His 395 400 405 Gly Pro Gly Pro Ile Phe Val Lys Asn Ile Leu Pro Lys Gly Ala 410 415 420 Ala Ile Lys Asp Gly Arg Leu Gln Ser Gly Asp Arg Ile Leu Glu 425 430 435 Val Asn Gly Arg Asp Val Thr Gly Arg Thr Gln Glu Glu Leu Val 440 445 450 Ala Met Leu Arg Ser Thr Lys Gln Gly Glu Thr Ala Ser Leu Val 455 460 465 Ile Ala Arg Gln Glu Gly His Phe Leu Pro Arg Glu Leu Lys Gly 470 475 480 Glu Pro Asp Cys Cys Ala Leu Ser Leu Glu Thr Ser Glu Gln Leu 485 490 495 Thr Phe Glu Ile Pro Leu Asn Asp Ser Gly Ser Ala Gly Leu Gly 500 505 510 Val Ser Leu Lys Gly Asn Lys Ser Arg Glu Thr Gly Thr Asp Leu 515 520 525 Gly Ile Phe Ile Lys Ser Ile Ile His Gly Gly Ala Ala Phe Lys 530 535 540 Asp Gly Arg Leu Arg Met Asn Asp Gln Leu Ile Ala Val Asn Gly 545 550 555 Glu Ser Leu Leu Gly Lys Ser Asn His Glu Ala Met Glu Thr Leu 560 565 570 Arg Arg Pro Met Ser Met Glu Gly Asn Ile Arg Gly Met Ile Gln 575 580 585 Leu Val Ile Leu Arg Arg Pro Glu Arg Pro Met Glu Asp Pro Ala 590 595 600 Glu Cys Gly Ala Phe Ser Lys Pro Cys Phe Glu Asn Cys Gln Asn 605 610 615 Ala Val Thr Thr Ser Arg Arg Asn Asp Asn Ser Ile Leu His Pro 620 625 630 Leu Gly Thr Cys Ser Pro Gln Asp Lys Gln Lys Gly Leu Leu Leu 635 640 645 Pro Asn Asp Gly Trp Ala Glu Ser Glu Val Pro Pro Ser Pro Thr 650 655 660 Pro His Ser Ala Leu Gly Leu Gly Leu Glu Asp Tyr Ser His Ser 665 670 675 Ser Gly Val Asp Ser Ala Val Tyr Phe Pro Asp Gln His Ile Asn 680 685 690 Phe Arg Ser Val Thr Pro Ala Arg Gln Pro Glu Ser Ile Asn Leu 695 700 705 Lys Ala Ser Lys Ser Met Asp Leu Val Pro Asp Glu Ser Lys Val 710 715 720 His Ser Leu Ala Gly Gln Lys Ser Glu Ser Pro Ser Lys Asp Phe 725 730 735 Gly Pro Thr Leu Gly Leu Lys Lys Ser Ser Ser Leu Glu Ser Leu 740 745 750 Gln Thr Ala Val Ala Glu Val Arg Lys Asn Asp Leu Pro Phe His 755 760 765 Arg Pro Arg Pro His Met Val Arg Gly Arg Gly Cys Asn Glu Ser 770 775 780 Phe Arg Ala Ala Ile Asp Lys Ser Tyr Asp Gly Pro Glu Glu Ile 785 790 795 Glu Ala Asp Gly Leu Ser Asp Lys Ser Ser His Ser Gly Gln Gly 800 805 810 Ala Leu Asn Cys Glu Ser Ala Pro Gln Gly Asn Ser Glu Leu Glu 815 820 825 Asp Met Glu Asn Lys Ala Arg Lys Val Lys Lys Thr Lys Glu Lys 830 835 840 Glu Lys Lys Lys Glu Lys Gly Lys Leu Lys Val Lys Glu Lys Lys 845 850 855 Arg Lys Glu Glu Asn Glu Asp Pro Glu Arg Lys Ile Lys Lys Lys 860 865 870 Gly Phe Gly Ala Met Leu Arg Tyr Gly Pro Ala Leu Lys Ala Lys 875 880 885 Leu Val Leu Ile Leu Ser Leu Leu Lys Lys Ala His Ala Phe Pro 890 895 900 Arg Leu Gln Pro Asn Ala Tyr Gly Ser Gln Phe Cys Ala Arg Ser 905 910 915 Leu Ser Ala Glu Ala Glu Glu Leu Phe Gly Glu Ser Tyr Ser Asp 920 925 930 Asp Arg Thr Leu Ser 935 2 1356 PRT Homo sapiens misc_feature Incyte ID No 2582063CD1 2 Met Lys Val Thr Val Cys Phe Gly Arg Thr Arg Val Val Val Pro 1 5 10 15 Cys Gly Asp Gly His Met Lys Val Phe Ser Leu Ile Gln Gln Ala 20 25 30 Val Thr Arg Tyr Arg Lys Ala Ile Ala Lys Asp Pro Asn Tyr Trp 35 40 45 Ile Gln Val His Arg Leu Glu His Gly Asp Gly Gly Ile Leu Asp 50 55 60 Leu Asp Asp Ile Leu Cys Asp Val Ala Asp Asp Lys Asp Arg Leu 65 70 75 Val Ala Val Phe Asp Glu Gln Asp Pro His His Gly Gly Asp Gly 80 85 90 Thr Ser Ala Ser Ser Thr Gly Thr Gln Ser Pro Glu Ile Phe Gly 95 100 105 Ser Glu Leu Gly Thr Asn Asn Val Ser Ala Phe Gln Pro Tyr Gln 110 115 120 Ala Thr Ser Glu Ile Glu Val Thr Pro Ser Val Leu Arg Ala Asn 125 130 135 Met Pro Leu His Val Arg Arg Ser Ser Asp Pro Ala Leu Ile Gly 140 145 150 Leu Ser Thr Ser Val Ser Asp Ser Asn Phe Ser Ser Glu Glu Pro 155 160 165 Ser Arg Lys Asn Pro Thr Arg Trp Ser Thr Thr Ala Gly Phe Leu 170 175 180 Lys Gln Asn Thr Ala Gly Ser Pro Lys Thr Cys Asp Arg Lys Lys 185 190 195 Asp Glu Asn Tyr Arg Ser Leu Pro Arg Asp Thr Ser Asn Trp Ser 200 205 210 Asn Gln Phe Gln Arg Asp Asn Ala Arg Ser Ser Leu Ser Ala Ser 215 220 225 His Pro Met Val Gly Lys Trp Leu Glu Lys Gln Glu Gln Asp Glu 230 235 240 Asp Gly Thr Glu Glu Asp Asn Ser Arg Val Glu Pro Val Gly His 245 250 255 Ala Asp Thr Gly Leu Glu His Ile Pro Asn Phe Ser Leu Asp Asp 260 265 270 Met Val Lys Leu Val Glu Val Pro Asn Asp Gly Gly Pro Leu Gly 275 280 285 Ile His Val Val Pro Phe Ser Ala Arg Gly Gly Arg Thr Leu Gly 290 295 300 Leu Leu Val Lys Arg Leu Glu Lys Gly Gly Lys Ala Glu His Glu 305 310 315 Asn Leu Phe Arg Glu Asn Asp Cys Ile Val Arg Ile Asn Asp Gly 320 325 330 Asp Leu Arg Asn Arg Arg Phe Glu Gln Ala Gln His Met Phe Arg 335 340 345 Gln Ala Met Arg Thr Pro Ile Ile Trp Phe His Val Val Pro Ala 350 355 360 Ala Asn Lys Glu Gln Tyr Glu Gln Leu Ser Gln Ser Glu Lys Asn 365 370 375 Asn Tyr Tyr Ser Ser Arg Phe Ser Pro Asp Ser Gln Tyr Ile Asp 380 385 390 Asn Arg Ser Val Asn Ser Ala Gly Leu His Thr Val Gln Arg Ala 395 400 405 Pro Arg Leu Asn His Pro Pro Glu Gln Ile Asp Ser His Ser Arg 410 415 420 Leu Pro His Ser Ala His Pro Ser Gly Lys Pro Pro Ser Ala Pro 425 430 435 Ala Ser Ala Pro Gln Asn Val Phe Ser Thr Thr Val Ser Ser Gly 440 445 450 Tyr Asn Thr Lys Lys Ile Gly Lys Arg Leu Asn Ile Gln Leu Lys 455 460 465 Lys Gly Thr Glu Gly Leu Gly Phe Ser Ile Thr Ser Arg Asp Val 470 475 480 Thr Ile Gly Gly Ser Ala Pro Ile Tyr Val Lys Asn Ile Leu Pro 485 490 495 Arg Gly Ala Ala Ile Gln Asp Gly Arg Leu Lys Ala Gly Asp Arg 500 505 510 Leu Ile Glu Val Asn Gly Val Asp Leu Val Gly Lys Ser Gln Glu 515 520 525 Glu Val Val Ser Leu Leu Arg Ser Thr Lys Met Glu Gly Thr Val 530 535 540 Ser Leu Leu Val Phe Arg Gln Glu Asp Ala Phe His Pro Arg Glu 545 550 555 Leu Asn Ala Glu Pro Ser Gln Met Gln Ile Pro Lys Glu Thr Lys 560 565 570 Ala Glu Asp Glu Asp Ile Val Leu Thr Pro Asp Gly Thr Arg Glu 575 580 585 Phe Leu Thr Phe Glu Val Pro Leu Asn Asp Ser Gly Ser Ala Gly 590 595 600 Leu Gly Val Ser Val Lys Gly Asn Arg Ser Lys Glu Asn His Ala 605 610 615 Asp Leu Gly Ile Phe Val Lys Ser Ile Ile Asn Gly Gly Ala Ala 620 625 630 Ser Lys Asp Gly Arg Leu Arg Val Asn Asp Gln Leu Ile Ala Val 635 640 645 Asn Gly Glu Ser Leu Leu Gly Lys Thr Asn Gln Asp Ala Met Glu 650 655 660 Thr Leu Arg Arg Ser Met Ser Thr Glu Gly Asn Lys Arg Gly Met 665 670 675 Ile Gln Leu Ile Val Ala Arg Arg Ile Ser Lys Cys Asn Glu Leu 680 685 690 Lys Ser Pro Gly Ser Pro Pro Gly Pro Glu Leu Pro Ile Glu Thr 695 700 705 Ala Leu Asp Asp Arg Glu Arg Arg Ile Ser His Ser Leu Tyr Ser 710 715 720 Gly Ile Glu Gly Leu Asp Glu Ser Pro Ser Arg Asn Ala Ala Leu 725 730 735 Ser Arg Ile Met Gly Glu Ser Gly Lys Tyr Gln Leu Ser Pro Thr 740 745 750 Val Asn Met Pro Gln Asp Asp Thr Val Ile Ile Glu Asp Asp Arg 755 760 765 Leu Pro Val Leu Pro Pro His Leu Ser Asp Gln Ser Ser Ser Ser 770 775 780 Ser His Asp Asp Val Gly Phe Val Thr Ala Asp Ala Gly Thr Trp 785 790 795 Ala Lys Ala Ala Ile Ser Asp Ser Ala Asp Cys Ser Leu Ser Pro 800 805 810 Asp Val Asp Pro Val Leu Ala Phe Gln Arg Glu Gly Phe Gly Arg 815 820 825 Gln Ser Met Ser Glu Lys Arg Thr Lys Gln Phe Ser Asp Ala Ser 830 835 840 Gln Leu Asp Phe Val Lys Thr Arg Lys Ser Lys Ser Met Asp Leu 845 850 855 Gly Ile Ala Asp Glu Thr Lys Leu Asn Thr Val Asp Asp Gln Lys 860 865 870 Ala Gly Ser Pro Ser Arg Asp Val Gly Pro Ser Leu Gly Leu Lys 875 880 885 Lys Ser Ser Ser Leu Glu Ser Leu Gln Thr Ala Val Ala Glu Val 890 895 900 Thr Leu Asn Gly Asp Ile Pro Phe His Arg Pro Arg Pro Arg Ile 905 910 915 Ile Arg Gly Arg Gly Cys Asn Glu Ser Phe Arg Ala Ala Ile Asp 920 925 930 Lys Ser Tyr Asp Lys Pro Ala Val Asp Asp Asp Asp Glu Gly Met 935 940 945 Glu Thr Leu Glu Glu Asp Thr Glu Glu Ser Ser Arg Ser Gly Arg 950 955 960 Glu Ser Val Ser Thr Ala Ser Asp Gln Pro Ser His Ser Leu Glu 965 970 975 Arg Gln Met Asn Gly Asn Gln Glu Lys Gly Asp Lys Thr Asp Arg 980 985 990 Lys Lys Asp Lys Thr Gly Lys Glu Lys Lys Lys Asp Arg Asp Lys 995 1000 1005 Glu Lys Asp Lys Met Lys Ala Lys Lys Gly Met Leu Lys Gly Leu 1010 1015 1020 Gly Asp Met Phe Arg Phe Gly Lys His Arg Lys Asp Asp Lys Ile 1025 1030 1035 Glu Lys Thr Gly Lys Ile Lys Ile Gln Glu Ser Phe Thr Ser Glu 1040 1045 1050 Glu Glu Arg Ile Arg Met Lys Gln Glu Gln Glu Arg Ile Gln Ala 1055 1060 1065 Lys Thr Arg Glu Phe Arg Glu Arg Gln Ala Arg Glu Arg Asp Tyr 1070 1075 1080 Ala Glu Ile Gln Asp Phe His Arg Thr Phe Gly Cys Asp Asp Glu 1085 1090 1095 Leu Met Tyr Gly Gly Val Ser Ser Tyr Glu Gly Ser Met Ala Leu 1100 1105 1110 Asn Ala Arg Pro Gln Ser Pro Arg Glu Gly His Met Met Asp Ala 1115 1120 1125 Leu Tyr Ala Gln Val Lys Lys Pro Arg Asn Ser Lys Pro Ser Pro 1130 1135 1140 Val Asp Ser Asn Arg Ser Thr Pro Ser Asn His Asp Arg Ile Gln 1145 1150 1155 Arg Leu Arg Gln Glu Phe Gln Gln Ala Lys Gln Asp Glu Asp Val 1160 1165 1170 Glu Asp Arg Arg Arg Thr Tyr Ser Phe Glu Gln Pro Trp Pro Asn 1175 1180 1185 Ala Arg Pro Ala Thr Gln Ser Gly Arg His Ser Val Ser Val Glu 1190 1195 1200 Val Gln Met Gln Arg Gln Arg Gln Glu Glu Arg Glu Ser Ser Gln 1205 1210 1215 Gln Ala Gln Arg Gln Tyr Ser Ser Leu Pro Arg Gln Ser Arg Lys 1220 1225 1230 Asn Ala Ser Ser Val Ser Gln Asp Ser Trp Glu Gln Asn Tyr Ser 1235 1240 1245 Pro Gly Glu Gly Phe Gln Ser Ala Lys Glu Asn Pro Arg Tyr Ser 1250 1255 1260 Ser Tyr Gln Gly Ser Arg Asn Gly Tyr Leu Gly Gly His Gly Phe 1265 1270 1275 Asn Ala Arg Val Met Leu Glu Thr Gln Glu Leu Leu Arg Gln Glu 1280 1285 1290 Gln Arg Arg Lys Glu Gln Gln Met Lys Lys Gln Pro Pro Ser Glu 1295 1300 1305 Gly Pro Ser Asn Tyr Asp Ser Tyr Lys Lys Val Gln Asp Pro Ser 1310 1315 1320 Tyr Ala Pro Pro Lys Gly Pro Phe Arg Gln Asp Val Pro Pro Ser 1325 1330 1335 Pro Ser Gln Val Ala Arg Leu Asn Arg Leu Gln Thr Pro Glu Lys 1340 1345 1350 Gly Arg Pro Phe Tyr Ser 1355 3 2968 DNA Homo sapiens misc_feature Incyte ID No 1555118CB1 3 aaggggcgct gccgcgagcc tccgggcctc agggtgttcc ggggagcggc gccccgggtc 60 tctgggccca cccgccccgg gcgtcctccg agagtggggg ctgcgcccgc ggggtcagac 120 acctgttcgg cccggcccgg cgtggtcgcc gggggccagg atgaaagtga ccgtgtgctt 180 cggcaggacg ggcatcgtgg tgccctgcaa ggagggccag ctgcgcgtcg gcgagctcac 240 ccagcaggcg ctgcagcggt acctgaagac ccgggagaag ggtcctggtt actgggtgaa 300 gattcatcac ttagaatata cagatggagg aatcctggat ccagatgatg tcttggcaga 360 tgttgttgaa gataaagaca agctgattgc tgtgtttgaa gaacaagaac cactccacaa 420 gattgagagc cccagtggaa accctgcaga tcggcagagc ccagatgctt ttgagacaga 480 agtggccgcc caactggctg catttaagcc aattggtggg gagattgaag taaccccttc 540 tgctctaaaa ctaggcactc cactgctggt gaggagaagc agtgacccag tgccaggccc 600 acctgctgat acccagccaa gcgcttcaca ccctggtggc cagagtctga aactggttgt 660 tccagattcc acgcagaact tggaagacag agaagttttg aatggtgtac agacagaact 720 actaacttcg ccaagaacta aggacacatt gagtgatatg acaagaacag tggagatttc 780 tggggaagga ggcccattgg gaatacatgt agtgcccttc ttttcatctc tgagtggaag 840 gattctagga ctcttcatcc gaggcattga agacaacagc aggtccaagc gggagggact 900 atttcacgaa aatgaatgta ttgtaaaaat caacaatgtg gatctcgtag acaaaacctt 960 tgctcaggct caagatgtct tccgccaggc aatgaaatct ccaagtgtgc tcctccacgt 1020 gcttcctcca caaaaccgtg aacagtatga aaagtcagtc attggctctc ttaacatttt 1080 tggtaataat gatggcgttt tgaaaaccaa agtgccgcct cctgtccatg gaaaatcggg 1140 actaaagaca gcaaatctca caggaaccga tagtcctgaa acagatgcat cagcttccct 1200 gcaacaaaac aagagtcccc gagtaccaag gctgggagga aaaccatcct ctccctcact 1260 ctcgcctctc atgggatttg gcagcaataa aaatgcaaag aaaattaaga ttgacctaaa 1320 gaaaggccct gaaggacttg gtttcactgt ggttaccaga gactcttcca tacatggtcc 1380 cggtcccatt tttgtaaaaa acattttacc aaagggagca gcaataaaag atggccgcct 1440 acaatcaggg gacagaattt tggaggtaaa tgggagagat gtcaccggac gaacccagga 1500 agagcttgtg gccatgctca ggagcaccaa gcagggggag acagcatcgc tggtcattgc 1560 ccgccaagaa ggacattttc tgccccgaga gttgaaagga gaacctgact gctgtgcact 1620 ctctctggag acaagcgagc agctcacctt tgagatcccc ctgaatgatt caggttctgc 1680 tggcctcggg gtgagcttaa aagggaacaa atccagagaa actggaacag acttggggat 1740 ttttatcaaa tccatcattc atggaggcgc tgcttttaag gatggtcgtc tgcgaatgaa 1800 tgaccagctg attgcagtta atggggaatc tcttttggga aagtccaacc acgaagctat 1860 ggaaacactt aggcggccaa tgtccatgga gggaaacatc cgagggatga tccagttggt 1920 gattctgagg aggccagaga gaccaatgga ggatcctgca gagtgtgggg cattttccaa 1980 gccatgcttt gagaactgtc aaaatgctgt aaccacctct aggcgaaatg ataatagtat 2040 cctgcatcca cttggcactt gcagtccaca agacaaacag aaaggtctat tgctgcccaa 2100 tgacggatgg gccgagagtg aagttccacc ttctccaaca ccacattctg ctctgggatt 2160 gggcctcgaa gattacagcc acagctctgg ggtggattca gcagtatatt ttccagatca 2220 gcacatcaac ttcagatctg tgacaccggc caggcagcct gaatcaatta atttgaaagc 2280 ctcgaagagc atggaccttg tgccagatga aagcaaggtt cactcattgg ctggacaaaa 2340 atcggaatct ccaagcaaag attttggtcc aactctgggt ttgaaaaagt ccagctcctt 2400 ggagagtctg cagactgcag tggccgaggt caggaagaat gaccttccct ttcacaggcc 2460 ccggccgcac atggttcgag gccgaggctg caatgagagc tttagagcag ccattgacaa 2520 atcctacgat ggacctgaag aaatagaagc tgacggtctg tctgataaga gctctcactc 2580 tggccaagga gctctgaatt gtgagtctgc ccctcagggg aattcggagc tagaggacat 2640 ggaaaataaa gccaggaaag tcaaaaaaac gaaagagaag gagaagaaaa aggaaaaggg 2700 caaattgaaa gtcaaggaga aaaagcgcaa agaggagaat gaagatccag aaaggaaaat 2760 aaagaagaag ggcttcggcg ccatgctgag gtatgggcct gctttgaagg caaagttggt 2820 tctcattttg tctctcctga aaaaagcgca cgcttttcct cgtcttcagc caaatgcata 2880 cggctctcaa ttctgtgctc gttctctttc tgcagaggca gaggagcttt ttggggaaag 2940 ttacagtgat gacaggacac tgtcttaa 2968 4 194 DNA Homo sapiens misc_feature Incyte ID No 1555118H1 4 caacaccaca ttctgctctg ggattgggcc tcgaagatta cagccacagc tctggggtgg 60 attcagcagt atattttcca gatcagcaca tcaacttcag atctgtgaca ccggccaggc 120 agcctgaatc aattaatttg aaagcctcga agagcatgga ccttgtgcca gatgaaagca 180 aggttcactc attg 194 5 533 DNA Homo sapiens misc_feature Incyte ID No 7227391H1 5 ggaatctgga acaaccagtt tcagactctg gccaccaggt gtgaagcgct tggctgggta 60 tcagcaggtg ggcctggcac tgggtcactg cttctcctca ccagcagtgg agtgcctagt 120 tttagagcag aaggggttac ttcaatctcc ccaccaattg gcttaaatgc agccagttgg 180 gcggccactt ctgtctcaaa agcatctggg ctctgccgat ctgcagggtt tccactgggg 240 ctctcaatct tgtggagtgg ttcttgttct tcaaacacag caatcagctt gtctttatct 300 tcaacaacat ctgccaagac atcatctgga tccaggattc ctccatctgt atattctaag 360 tgatgaatct tcacccagta accaggaccc ttctcccggg tcttcaggta ccgctgcagc 420 gcctgctggg tgagctcgcc gacgcgcagc tggccctcct tgcagggcac cacgatgccc 480 gtcctgccga agcacacggt cactttcatc ctggcccccg gcgaccacgc cgg 533 6 588 DNA Homo sapiens misc_feature Incyte ID No 70158486V1 6 ccatggacag gaggcggcac tttggttttc aaaacgccat cattattacc aaaaatgtta 60 agagagccaa tgactgactt ttcatactgt tcacggtttt gtggaggaag cacgtggagg 120 agcacacttg gagatttcat tgcctggcgg aagacatctt gagcctgagc aaaggttttg 180 tctacgagat ccacattgtt gatttttaca atacattcat tttcgtgaaa tagtccctcc 240 cgcttggacc tgctgttgtc ttcaatgcct cggatgaaga gtcctagaat ccttccactc 300 agagatgaaa agaagggcac tacatgtatt cccaatgggc ctccttcccc agaaatctcc 360 actgttcttg tcatatcact caatgtgtcc ttagttcttg gcgaagttag tagttctgtc 420 tgtacaccat tcaaaacttc tctgtcttcc aagttctgcg tggaatctgg aacaaccagt 480 ttcagactct ggccaccagg gtgtgaagcg cttggctggg tatcagcagg tgggcctggc 540 actggggtca tgcttctcat cacaagcagt ggagtgccta gttttaga 588 7 481 DNA Homo sapiens misc_feature Incyte ID No 70162686V1 7 gaccatgtat ggaagagtct ctggtaacca cagtgaaacc aagtccttca gggcctttct 60 ttaggtcaat cttaattttc tttgcatttt tattgctgcc aaatcccatg agaggcgaga 120 gtgagggaga ggatggtttt cctcccagcc ttggtactcg gggactcttg ttttgttgca 180 gggaagctga tgcgtctgtt tcaggactat cggttcctgt gagatttgct gtctttagtc 240 ccgattttcc atggacagga ggcggcactt tggttttcaa aacgccatca ttattaccaa 300 aaatgttaag agagccaatg actgactttt catactgttc acggttttgt ggaggaaagc 360 acgtggagga gcacacttgg agatttcatt gcctggcgga agacatcttg agcctgagca 420 aaggttttgt ctacgagatc cacattgttg atttttacaa tacattcatt ttcgggaaat 480 a 481 8 355 DNA Homo sapiens misc_feature Incyte ID No 70151326V1 8 accagagact cttccataca tggtcccggt cccatttttg taaaaaacat tttaccgaag 60 ggagcagcaa taaaagatgg ccgcctacaa tcaggggaca gaattttgga ggtaaatggg 120 agagatgtca ctggacgaac ccaggaagag cttgtggcca tgctcaggag caccaagcag 180 ggggagacag catcgctggt cattgcccgc caagaaggac attttctgcc ccgagagttg 240 aaaggagaac ctgactgctg tgcactctct ctggagacaa gcgagcagct cacctttgag 300 aatccccctg gatgattcag gttctgctgg cctcggggtg agcttaaaag ggaac 355 9 417 DNA Homo sapiens misc_feature Incyte ID No 70154198V1 9 gctgtaatct tcgaggccca atcccagagc agaatgtggt gttggagaag gtggaacttc 60 actctcggcc catccgtcat tgggcagcaa tagacctttc tgtttgtctt gtggactgca 120 agtgccaagt ggatgcagga tactattatc atttcgccta gaggtggtta cagcattttg 180 acagttctca aagcatggct tggaaaatgc cccacactct gcaggatcct ccattggtct 240 ctctggcctc ctcagaatca ccaactggat catccctcgg atggttccct ccatggacat 300 tggccgccta agtgtttcca tagcttcgtg gttggacttc ccaaaagaga ttccccatta 360 cggcaatcac tggtcattca tttgcagacg accatccttt aaagcagcgc cttcacg 417 10 537 DNA Homo sapiens misc_feature Incyte ID No 2084238T6 10 ccttgacttt caatttgccc ttttcctttt tcttctcctt ctctttcgtt tttttgactt 60 tcctggcttt attttccatg tcctctagct ccgaattccc ctgaggggca gactcacaat 120 tcagagctcc ttggccagag tgagagctct tatcagacag accgtcagct tctatttctt 180 caggtccatc gtaggatttg tcaatggctg ctctaaagct ctcattgcag cctcggcctc 240 gaaccatgtg cggccggggc ctgtgaaagg gaaggtcatt cttcctgacc tcggccactg 300 cagtctgcag actctccaag gagctggact ttttcaaacc cagagttgga ccaaaatctt 360 tgcttggaga ttccgatttt tgtccagcca atgagtgaac cttgctttca tctggcacaa 420 ggtccatgct cttcgaggct ttcaaattaa ttgattcagg ctgctggccg gtgtcacaga 480 tctgaagttg atgtgctgat ctggaaaata tactgctgaa tccaccccag agtgtgg 537 11 498 DNA Homo sapiens misc_feature Incyte ID No 70155923V1 11 cctttctgga tcttcattct cctcttggcg ctttttctcc ttgactttca atttgccctt 60 tcccttttcc ttctccttct ctttcgtttt tttgactttc ctggcttaat ttcccatgtc 120 ctctagctcc gaattcccct gaggggcaga ctcacaattc agagctcctt ggccagagtg 180 aaagctctta tcagacagac cgtcagcttc aatttcttaa ggtccatcgt aggatttgca 240 aatggctgct ctaaagctct cattgaagcc tcggcctcga accatgtgcg gccggggcct 300 ttgaaaggga aggtcattct tcctgacctc ggccactgca gtttgcagac tctccaagga 360 gctggacttt tctaaaccca gagttggccc aaaatctttg cttggaaatt ccgatttttg 420 tccagccaat gagtgaacct tgctttcatc tggcaccagg tccatgccct tcggggcttt 480 caaattaatt ggattcag 498 12 498 DNA Rattus norvegicus misc_feature Incyte ID No 702457609T1 12 ttggctttat tttccacaca catctagctc agggtttccc tgaggggcag actcacaatt 60 cagagctgtg tggcccgagt gagagctctt atcagacaga ccatcagctt ctgcctcttc 120 tggcccgtcg taagatttgt caatggctgc tcgagaagct ctcattgcag ccccggccgt 180 cgcaccatgt gtggtctggg tctgtggaag ggcagatcat tcttcctgac ttcagccaca 240 gcagtctgta ggctctccaa ggaactggac tttttcaggc ccagggttgg accaaaatct 300 ttgcctggag agtctgacct gcgatcagcc agtgactgga ctttgctttc gtctggcaca 360 aggtccatgc tcttggaggc tttcaggtta attgattcgg gctgcccgac gtggtgtcac 420 aattctgaaa ttgacatgtt gatctggaaa atatcctgtg gaatccactc tagagctgtg 480 actgaagtct tcaaggcc 498 13 460 DNA Rattus norvegicus misc_feature Incyte ID No 702458746T1 13 gtgtggcccg aagtgagagc tcttatcaga cagaccatca gcttctgcct cttctggccc 60 gtcgtaagat ttgtcaatgg ctgctcggaa gctctcattg cagccccggc ctcgcaccat 120 gtgtggtctg ggtctgtgga agggcagatc attcttcctg acttcagcca cagcagtctg 180 taggctctcc aaggaactgg actttttcag gcccagggtt ggaccaaaat ctttgcctgg 240 agagtctgac ctgcgatcag ccagtgactg gactttgctt tcgtctggca caaggtccat 300 gctcttggag gctttcaggt taattgattc gggctgcccg actggtgtca caattctgaa 360 attgacatgt tgatctggaa aatatcctgt ggaatccact ctagagctgt gactgaagtc 420 ttcaaggccc cattccagac tgggatgtgg tggcggggac 460 14 245 DNA Rattus norvegicus misc_feature Incyte ID No 701335936H1 14 aaaggccccg aaggacttgg attcactgtg ggaactagag attcttggat acatggacct 60 ggtcccattt ttgtaaaaaa tatcctacca aagggagcag cagtaaagga tggccgccta 120 caatcaggag acagaatttt agaggtaaat ggcagagatg tcacaggaag aacccaggaa 180 gaacttgtgg ccatgctgag gagcactaag cagggagaga cggtatcact ggtcattgcc 240 cgcca 245 15 260 DNA Rattus norvegicus misc_feature Incyte ID No 700639694H1 15 attttggtcc aaccctgggc ctgaaaaagt ccagttcctt ggagagccta cagactgctg 60 tggctgaagt caggaagaat gatctgccct tccacagacc cagaccacac atggtgcgag 120 gccggggctg caatgagagc ttccgagcag ccattgacaa atcttacgac gggccagaag 180 aggcagaagc tgatggtctg tctgataaga gctctcgctc gggccacaca gctctgaatt 240 gtgagtctgc ccctcaggga 260 16 211 DNA Rattus norvegicus misc_feature Incyte ID No 700639694F6 16 gatctgccct tccacagacc cagaccacac atggtgcgag gccggggctg caatgagagc 60 ttccgagcag ccattgacaa atcttacgac gggccagaag aggcagaagc tgatggtctg 120 tctgataaga gctctcgctc gggccacaca gctctgaatt gtgagtctgc ccctcaggga 180 aaccctgagc tagatgatgt ggaaaataaa g 211 17 276 DNA Rattus norvegicus misc_feature Incyte ID No 701191467H1 17 ctatagtcca caagataaaa ggaaagacct attgcttccc agtgatgggt gggctgagaa 60 tgaagtaccg ccgtccccgc caccacatcc cagtctggaa tggggccttg aagacttcag 120 tcacagctct agagtggatt cacaggatat tttccagatc accatgtcaa tttcagaatt 180 gtgacaccag tcgggcagcc cgaatcaatt aacctgaaag cctccaggag catggacctt 240 gtgccagacg aaagcaaagt ccagtcactg gctgat 276 18 555 DNA Canis familiaris misc_feature Incyte ID No 702771158H1 18 aagatgttcc accttcccct cctcagcacc acagagtggc ctaccaggaa atgggtagac 60 caggccccga gggggcagcc cagaccagta cccctaccgc gcccaggatc ccaggcagaa 120 gaaccccatg actgcagccg tgtagctgaa taccaccgag ctcagcccag cccagaaggg 180 cgacatctga catcaccttc gccctcccta gactcttaag gccttcctcc tgtccagaag 240 tctccatggt acagataggt tttgctcacc gaggttgcaa cacttgactg ctgaccagag 300 gggaaaagga gaggacagga gggtgggaga gaaaggacag gagtcacaaa gacagcactg 360 cctgggattt gaaatatgtt tagaatctct cagttgaggg caagtggcag ttgagcaggc 420 aaataccaat ggagacatag cacacggggc ctccctggcg tacaccattc ataacttctc 480 catcttctaa gttctgtgtg gaatctagaa taacaggttt cagactctgg ccactggggt 540 gtgaagcact tggct 555 19 257 DNA Mus musculus misc_feature Incyte ID No 701266650H1 19 gaaaaatgca aaaaaaatta agattgacct aaagaaaggc cctgagggac ttggattcac 60 tgtggaacca gagattcttc tatacatggt cctggtccca tttctgtaaa aaacatctta 120 ccaaaaggag cagcagtaaa ggatggccgc ctacaatcag gggacagaat tttggaggta 180 aatggcagag atgttacagg aagaacccaa gaagaactcg tggccatgtt aaggagcacc 240 aagcagggag agacagt 257 20 5689 DNA Homo sapiens misc_feature Incyte ID No 2582063CB1 20 atgaaagtga ccgtgtgctt cggacggacc cgggtggtcg tgccgtgcgg ggacggccac 60 atgaaagttt tcagcctcat ccagcaggcg gtgacccgct accggaaggc catcgccaag 120 gatccaaact actggataca ggtgcatcgc ttggaacatg gagatggagg aatactagac 180 cttgatgaca ttctttgtga tgtagcagac gataaagaca gactggtagc agtgtttgat 240 gagcaggatc cacatcacgg aggtgatggc accagtgcca gttccacggg tacccagagc 300 ccagagatat ttggtagtga gcttggcacc aacaatgtct cagcctttca gccttaccaa 360 gcaacaagtg aaattgaggt cacaccttca gtccttcgag caaatatgcc tcttcatgtt 420 cgacgcagta gtgacccagc tctaattggc ctctccactt ctgtcagtga tagtaatttt 480 tcctctgaag agccttcaag gaaaaatccc acacgctggt caacaacagc tggcttcctc 540 aagcagaaca ctgctgggag tcctaaaacc tgcgacagga agaaagatga aaactacaga 600 agcctcccgc gggatactag taactggtct aaccaatttc agagagacaa tgctcgctcg 660 tctctgagtg ccagtcaccc aatggtgggc aagtggctgg agaaacaaga acaggatgag 720 gatgggacag aagaggataa cagtcgtgtt gaacctgttg gacatgctga cacgggtttg 780 gagcatatac ccaacttttc tctggatgat atggtaaagc tcgtagaagt ccccaacgat 840 ggagggcctc tgggaatcca tgtagtgcct ttcagtgctc gaggcggcag aaccctgggg 900 ttattagtaa aacgattgga gaaaggtggt aaagctgaac atgaaaatct ttttcgtgag 960 aatgattgca ttgtcaggat taatgatggc gaccttcgaa atagaagatt tgaacaagca 1020 caacatatgt ttcgccaagc catgcgtaca cccatcattt ggttccatgt ggttcctgca 1080 gcaaataaag agcagtatga acaactatcc caaagtgaga agaacaatta ctattcaagc 1140 cgttttagcc ctgacagcca gtatattgac aacaggagtg tgaacagtgc agggcttcac 1200 acggtgcaga gagcaccccg actgaaccac ccgcctgagc agatagactc tcactcaaga 1260 ctacctcata gcgcacaccc ctcgggaaaa ccaccatccg ctccagcctc ggcacctcag 1320 aatgtattta gtacgactgt aagcagtggt tataacacca aaaaaatagg caagaggctt 1380 aatatccagc ttaagaaagg tacagaaggt ttgggattca gcatcacttc cagagatgta 1440 acaataggtg gctcagctcc aatctatgtg aaaaacattc tcccccgggg ggcggccatt 1500 caggatggcc gacttaaggc aggagacaga cttatagagg taaatggagt agatttagtg 1560 ggcaaatccc aagaggaagt tgtttcgctg ttgagaagca ccaagatgga aggaactgtg 1620 agccttctgg tctttcgcca ggaagacgcc ttccacccaa gggaactgaa tgcagagcca 1680 agccagatgc agattccaaa agaaacgaaa gcagaagatg aggatattgt tcttacacct 1740 gatggcacca gggaatttct gacatttgaa gtcccactta atgattcagg atctgcaggc 1800 cttggtgtca gtgtcaaagg taaccggtca aaagagaacc acgcagattt gggaatcttt 1860 gtcaagtcca ttattaatgg aggagcagca tctaaagatg gaaggcttcg ggtgaatgat 1920 caactgatag cagtaaatgg agaatccctg ttgggcaaga caaaccaaga tgccatggaa 1980 accctaagaa ggtctatgtc tactgaaggc aataaacgag gaatgatcca gcttattgtt 2040 gcaaggagaa taagcaagtg caatgagctg aagtcacctg ggagcccccc tggacctgag 2100 ctgcccattg aaacagcgtt ggatgataga gaacgaagaa tttcccattc cctctacagt 2160 gggattgagg ggcttgatga atcgcccagc agaaatgctg ccctcagtag gataatgggt 2220 gagtcaggta aataccagct gtcccctaca gtgaatatgc cccaagatga cactgtcatt 2280 atagaagatg acaggttgcc agtgcttcct ccacatctct ctgaccagtc ctcttccagc 2340 tcccatgatg atgtggggtt tgtgacggca gatgctggta cttgggccaa ggctgcaatc 2400 agtgattcag ccgactgctc tttgagtcca gatgttgatc cagttcttgc ttttcaacga 2460 gaaggatttg gacgtcagag tatgtcagaa aaacgcacaa agcaattttc agatgccagt 2520 caattggatt tcgttaaaac acgaaaatca aaaagcatgg atttaggtat agctgacgag 2580 actaaactca atacagtgga tgaccagaaa gcaggttctc ccagcagaga tgtgggtcct 2640 tccctgggtc tgaagaagtc aagctcgttg gagagtctgc agaccgcagt tgccgaggtg 2700 actttgaatg gggatattcc tttccatcgt ccacggccgc ggataatcag aggcagggga 2760 tgcaatgaga gcttcagagc tgccatcgac aaatcttatg ataaacccgc ggtagatgat 2820 gatgatgaag gcatggagac cttggaagaa gacacagaag aaagttcaag atcagggaga 2880 gagtctgtat ccacagccag tgatcagcct tcccactctc tggagagaca aatgaatgga 2940 aaccaagaga aaggtgataa gactgataga aaaaaggata aaactggaaa agaaaagaag 3000 aaagatagag ataaggagaa ggataaaatg aaagccaaga agggaatgct gaagggcttg 3060 ggagacatgt tcaggtttgg caaacatcga aaagatgaca agattgagaa aacgggtaaa 3120 ataaaaatac aggaatcctt tacatcagaa gaggagagga tacgaatgaa gcaggagcag 3180 gagaggattc aagccaaaac tcgagaattt agggaacgac aagctcgaga gcgtgactat 3240 gctgaaattc aagattttca tcggacattt ggctgtgatg atgagttaat gtatggggga 3300 gtttcttctt atgaaggttc catggctctc aacgctagac ctcagagccc acgagaaggg 3360 catatgatgg atgctttgta tgcccaagtc aagaagccgc ggaattccaa accctcacct 3420 gtagacagta acagatcaac tcctagcaat catgatcgga tacagcgtct gaggcaagaa 3480 tttcagcaag caaagcaaga tgaagatgta gaagatcgtc ggcggaccta tagttttgag 3540 caaccctggc cgaacgcacg gccggcgacg cagagcgggc gacactcggt gtccgtggag 3600 gtgcagatgc agcggcagcg gcaggaggag cgcgagagct cccagcaggc ccagcgccag 3660 tacagctctc tgcctcggca aagcaggaaa aatgccagct cggtctccca ggactcttgg 3720 gagcagaact actcccctgg ggaaggcttc cagagtgcca aagagaaccc caggtactcc 3780 agctaccaag gctccaggaa cggctacctg ggaggacatg gcttcaacgc cagggtcatg 3840 ctggaaactc aggagctcct tcgccaggaa cagaggcgga aggagcagca gatgaagaag 3900 cagcctcctt ccgaggggcc cagcaactat gactcgtata agaaagtcca ggaccccagt 3960 tacgcccctc ccaaggggcc cttccggcaa gatgtgcccc cctccccttc tcaggttgcg 4020 aggctgaaca gacttcagac tcctgagaaa gggaggccct tctattcctg agcacgcaaa 4080 taacggatgc ttcatgtcgc gcaataaaag acattttcct atgaagactt gtattttggg 4140 agttttttta aaacctcgat ggtactatgg agtatttctg ttgttggtat cagtgccttt 4200 aagcggtgta ggcaaagaaa tggaaggcct taatgtcttt gccactatgt ctcaagtgtc 4260 tgtttcatgg aaggatttcc caccctgtga caatcatctg tttgaggtgt tcatatgctc 4320 tgcgcctctc cacagtacca ggaatctcgg ccctactcat gagttgtccg cggcttggtt 4380 gtaacatccc tgcaccactt gcagtgacaa attcacctga agtggaggat gacgtgcggc 4440 cctgtttctc cctctaagtt ctcttagcta tgggatgaca tcttagtctc tggtggagga 4500 aaagtgggcg acatacacca aaaattgggg ctttctggta cttcacagca cagccatttg 4560 tcgtactttg tcatcactgt ggttttctct ttcctttctc agctctttgt gacgggagag 4620 tcggtcatcc tattacagaa gctaagccat agtccaacat tgtttggtca ccatgggggt 4680 ccttttgtaa ctgccttatg actcaacatt accaataaag tgatgatcct ggtctgcgtt 4740 tatacatacg cttgttcggt cctgttcctg acacgtgggt tgagtcacca cagctctgtg 4800 tggggaacgt gggagacagg agtggctcct gccgggggaa gctgggcctg ccattggccc 4860 tgtgtctatc atgaggggag agctaagaaa gaaattctcc taggaagagc tcatggccca 4920 gtacatccta gtaattattt taattagttt ttgttctgac agcttgtcag gaagggcaca 4980 gaatgggaca gagataaacc agacagtcat tttgatctgc tctctacggt ttttcaagtc 5040 agaggcaatt gatgcttgtc taatgcatcc acacactgca tgtctgactg gcgatgccac 5100 gctcctaagt agttctgcca tgaaacataa aagacaaagg aaaagccgtt acacatcaca 5160 cagagaacat tttcgggtcc cacagcggtg gtggcaggaa gctcactctc gcgtcagtat 5220 tagagtgtgt gtgtgggtct cggggatctc ggtggctccc atcttccttc attgttctga 5280 acatcctgta ttgtaaacca tggctggggt gctaaagtgc ctgtgaatcc cgatgtggaa 5340 aaagctggag gtgaaagctc agcataccat gtatttactt taaaaacaga aaaaaagaca 5400 tgtatggata tgtctatttt ttttttattg gcacattgta tttttgtgtt gacttgtttt 5460 tagaaatgat gtgtccacac acgtacccgt gtctcttctg catttctgtg tcatggttct 5520 gtttcttaat cacgtgcggc ggtgtctaag tggtgttacc agtgtacgcg cagtgacctt 5580 ggatgacagt ggctctttct cacagcctcc cctgagctgt gagaaacagc tttctctgta 5640 catatgcaac tcctaataaa aggcatattt cttcctgtta aaaaaaaaa 5689 21 249 DNA Homo sapiens misc_feature Incyte ID No 2582063H1 21 cagcggtggt ggcaggaagc tcactctcgc gtcagtatta gagtgtgtgt gtgggtctcg 60 gggatctcgg tggctcccat cttccttcat tgttctgaac atcctgtatt gtaaaccatg 120 gctggggtgc taaagtgcct gtgaatcccg atgtggaaaa agctggaggt gaaagctcag 180 cataccatgt atttacttta aaaacagaaa aaaagacatg tatggatatg tctatttttt 240 ttttattgg 249 22 549 DNA Homo sapiens misc_feature Incyte ID No 7246093H1 22 cccgggtggt cgtgccgtgc ggggacggcc acatgaaagt tttcagcctc atccagcagg 60 cggtgacccg ctaccggaag gccatcgcca aggatccaaa ctactggata caggtgcatc 120 gcttggaaca tggagatgga ggaatactag accttgatga cattctttgt gatgtagcag 180 acgataaaga cagactggta gcagtgtttg atgagcagga tccacatcac ggaggtgatg 240 gcaccagtgc cagttccacg ggtacccaga gcccagagat atttggtagt gagcttggca 300 ccaacaatgt ctcagccttt cagccttacc aagcaacaag tgaaattgag gtcacacctt 360 cagtccttcg agcaaatatg cctcttcatg ttcgacgcag tagtgaccca gctctaattg 420 gcctctccac ttctgtcagt gatagtaatt tttcctctga agagccttca aggaaaaatc 480 ccacacgctg gtcaacaaca gctggcttcc tcaagcagaa cactgctggg agtcctaaaa 540 cctgcgaca 549 23 502 DNA Homo sapiens misc_feature Incyte ID No 7978420H1 23 ggggagccca gagatatttg gtagtgagct tggcaccaac aatgtctcag cctttcagcc 60 ttaccaagca acaagtgaaa ttgaggtcac accttcagtc cttcgagcaa atatgcctct 120 tcatgttcga cgcagtagtg acccagctct aattggcctc tccacttctg tcagtgatag 180 taatttttcc tctgaagagc cttcaaggaa aaatcccaca cgctggtcaa caacagctgg 240 cttcctcaag cagaacactg ctgggagtcc taaaacctgc gacaggaaga aagatgaaaa 300 ctacagaagc ctcccgcggg atactagtaa ctggtctaac caatttcaga gagacaatgc 360 tcgctcgtct ctgagtgcca gtcacccaat ggtgggcaag tggctggaga aacaagaaca 420 ggatgaggat gggacagaag aggataacag tcgtgttgaa cctgttggac atgctgacac 480 gggtttggag catataccca ac 502 24 611 DNA Homo sapiens misc_feature Incyte ID No 55040412H1 24 gctgtgcatg aagacgggac agaagaggat aacagtcgtg ttgaacctgt tggacatgct 60 gacacgggtt tggagcatat acccaacttt tctctggatg atatggtaaa gctcgcagaa 120 gtccccaacg atggagggcc tctgggaatc catgtagtgc ctttcagtgc tcgaggcggc 180 agaaccctgg ggttattagt aaaacgattg gagaaaggtg gtaaagctga acatgaaaat 240 ctttttcgtg agaatgattg cattgtcagg attaatgatg gcgaccttcg aaatagaaga 300 tttgaacaag cacaacatat gtttcgccaa gccatgcgta cacccatcat ttggttccat 360 gtggttcctg cagcaaataa agagcagtat gaacaactat cccaaagtga gaagaacaat 420 tactattcaa gccgttttag ccctgacagc cagtatattg acaacaggag tgtgaacagt 480 gcagggctgc acacggtgca gagagcaccc cgactgaacc acccgcctga gcagatagac 540 tctcactcaa gactacctca tagcgcacac ccctcgggaa aaccaccatc cgctccatcc 600 tcatggacag c 611 25 462 DNA Homo sapiens misc_feature Incyte ID No 2929484F6 25 gagcaccccg actgaaccac ccgcctgagc agatagactc tcactcaaga ctacctcata 60 gcgcacaccc ctcgggaaaa ccaccatccg ctccagcctc ggcacctcag aatgtattta 120 gtacgactgt aagcagtggt tataacacca aaaaaatagg caagaggctt aatatccagc 180 ttaagaaagg tacagaaggt ttgggattca gcatcacttc cagagatgta acaataggtg 240 gctcagctcc aatctatgtg aaaaacattc tcccccgggg ggcggccatt caggatggcc 300 gacttaaggc aggagacaga cttatagagg taaatggagt agatttagtg ggccaatccc 360 aagaggaagt tgtttcgctg ttgagaagca ccaagatgga aggantgtga gcttctggtc 420 tttcgccagg aagacgcttc nacccaaggg aactgaatgc ag 462 26 375 DNA Homo sapiens misc_feature Incyte ID No 5627320R8 26 acactgcgtg gttctctttt gaccggttac ctttgacact gacaccaagg cctgcagatc 60 ctgaatcatt aagtgggact tcaaatgtca gaaattccct ggtgccatca ggtgtaagaa 120 caatatcctc atcttctgct ttcgtttctt ttggaatctg catctggctt ggatctgcat 180 tcagttccct tgggtggaag gcgtcttcct ggcgaaagac cagaaggctc acagttcctt 240 ccatcttggt gcttctcaac agcgaaacaa cttcctcttg ggatttgccc actaaatcta 300 ctccatttac ctctataagt ctgtctcctg acttaagtcg gccatcctga atggccgccc 360 ccgggggaga atgtt 375 27 543 DNA Homo sapiens misc_feature Incyte ID No 3209128F6 27 cgccttccac ccaagggaac tgaaagcaga agatgaggat attgttctta cacctgatgg 60 caccaggaaa tttctgacat ttgaagtccc acttaatgat tcaggatctg caggccttgg 120 tgtcagtgtc aaaggtaacc ggtcaaaaga gaaccacgca gatttgggaa tctttgtcaa 180 gtccattatt aatggaggag cagcatctaa agatggaagg cttcgggtga atgatcaact 240 gatagcagta aatggagaat ccctgttggg caagacaaac caagatgcca tggaaaccct 300 aagaaggtct atgtctactg aaggcaataa acgaggaatg atccagctta ttgttgcaag 360 gagaataagc aagtgcaatg agctgaagtc acctgggagc ccccctggac ctgagctgcc 420 cattgaaaca gcgttggatg atagagaacg aagaatttcc cattccctct acagtgggat 480 tgaggggctt gatgaatcgg ccagcagaaa tgctggcctc agtaggataa tgggtgagtc 540 agg 543 28 220 DNA Homo sapiens misc_feature Incyte ID No 349248H1 28 aatatgcccc aagatgacac tgtcattata gaagatgaca ggttgccagt gcttcctcca 60 catctctctg accagtcctc ttccagctcc catgatgatg tggggtttgt gacggcagat 120 gctggtactt gggccaaggc tgcaatcagt gattcagccg actgctnttt gagtccagat 180 gttgatccag ttcttgcttt tcaacgagaa ggatttggac 220 29 613 DNA Homo sapiens misc_feature Incyte ID No 7019961H1 29 gtgcttcctc cacatctctc tgaccagtcc tcttccagct cccatgatga tgtggggttt 60 gtgacggcag atgctggtac ttgggccaag gctgcaatca gtgattcagc cgactgctct 120 ttgagtccag atgttgatcc agttcttgct tttcaacgag aaggatttgg acgtcagagt 180 atgtcagaaa aacgcacaaa gcaattttca gatgccagtc aattggattt cgttaaaaca 240 cgaaaatcaa aaagcatgga tttaggtata gctgacgaga ctaaactcaa tacagtggat 300 gaccagaaag caggttctcc cagcagagat gtgggtcctt ccctgggtct gaagaagtca 360 agctcgttgg agagtctgca gaccgcagtt gccgaggtga ctttgaatgg ggatattcct 420 ttccatcgtc cacggccgcg gataatcaga ggcaggggat gcaatgagag cttcagagct 480 gccatcgaca aatcttatga taaacccgcg gtagatgatg atgatgaagg catggagacc 540 ttggaagaag acacagaaga cagttcacga tcagggagag agtctgtatc cacagccagg 600 atcaggcttc cac 613 30 249 DNA Homo sapiens misc_feature Incyte ID No 6303175H2 30 tcgcgatcta gaacaaagaa aagaagaaag atagagataa ggagaaggat aaaatgatag 60 ccaagaaggg aatgctgaag ggcttgggag acatgttcag gtttggcaaa catcgaaaag 120 atgacaagat tgagaaaacg ggtaaaataa aaatacagga atcctttaca tcagaagagg 180 agaggatacg aatgaagcag gagcaggaga ggattcaagc caaaactcga gaatttaggg 240 aacgacaag 249 31 501 DNA Homo sapiens misc_feature Incyte ID No 2549906F6 31 aggagaagga taaaatgaaa gccaagaagg gaatgctgaa gggcttggga gacatgttca 60 ggtttggcaa acatcgaaaa gatgacaaga ttgagaaaac gggtaaaata aaaatacagg 120 aatcctttac atcagangag gagaggatac gaatgaagca ggancaggag aggattcaag 180 ccaaaactcg agaatttagg gaacgacaag ctcgagagcg tgactatgct gaaattcaag 240 attttcatcg gacatttggc tgtgatgatg agttaatgta tgggggagtt tcttcttatg 300 aaggttccat ggctctcaac gctagacctc agagcccacg agaagggcat atgatggatg 360 ctttgtatgc ccaagtcaag aagccgcgga attccaaacc ctcacctgta gacagtaaca 420 gatcaactcc tagcaatcat gatcggatac agcgtctgag gcnagaattt cagcaagcaa 480 agcaagatga agatgtagaa g 501 32 265 DNA Homo sapiens misc_feature Incyte ID No 1945452H1 32 gttccatggc tctcaacgct agacctcaga gcccacgaga agggcatatg atggatgctt 60 tgtatgccca agtcaagaag ccgcggaatt ccaaaccctc acctgtagac agtaacagat 120 caactcctag caatcatgat cggatacagc gtctgaggca agaatttcag caagcaaagc 180 aagatgaaga tgtagaagat cgtcggcgga cctatagttt tgagcaaccc tggccgaacg 240 cacggccggc gacgcagagc gggcg 265 33 469 DNA Homo sapiens misc_feature Incyte ID No 2549906T6 33 gttatttgcg tgctcaggaa tagaagggcc tccctttctc aggagtctga agtctgttca 60 gcctcgcaac ctgaganggg gaggggggca catcttgccg gaagggcccc ttgggagggg 120 cgtaactggg gtcctggact ttcttatacg agtcatagtt gctgggcccc tcggaaggag 180 gctgcttctt catnanctgc tccttccgcc tctgttcctg gcgaaggagc tcctgagttt 240 ccagcatgac cctggcgttg aagccatgtc ctcccaggta gccgttcctg gagccttggt 300 agctggagta cctggggttc tctttggcac tctggaagcc ttccccaggg gagtagttct 360 gctcccaaga gtcctgggag accgagctgg catttttcct gctttgccga ggcagagagc 420 tgtactggcg ctgggcctgt ngggagtctc gcgctcctcc tgccgntgc 469 34 558 DNA Homo sapiens misc_feature Incyte ID No 71009002V1 34 caggcccagc gccagtacag ctctctgcct cggcaaagca ggaaaaatgc cagctcggtc 60 tcccaggact cttgggagca gaactactcc cctggggaag gcttccagag tgccaaagag 120 aaccccaggt actccagcta ccaaggctcc aggaacggct acctgggagg acatggcttc 180 aacgccaggt catgctggaa actcaggagc tccttcgcca ggaacagagg cggaaggagc 240 agcagatgaa gaagcagcct ccttccgagg ggcccagcaa ctatgactcg tataagaaag 300 tccaggaccc cagttacgcc cctcccaagg ggcccttccg gcaagatgtg cccccctccc 360 cttctcaggt tgcgaggctg aacagacttc agactcctga gaaagggagg cccttctatt 420 cctgagcacg caaataacgg atgcttcatg tcgcgcaata aaagacattt tcctatgaag 480 acttgtattc cgggagtttt ttaaaaacct cgatggtact atggagtata ctggtcgtgg 540 tatcagtgcc tttaagcg 558 35 632 DNA Homo sapiens misc_feature Incyte ID No 71008521V1 35 ttccccacac agagctgtgg tgactcaacc cacgtgtcag gaacaggacc gaacaagcgt 60 atgtataaac gcagaccagg atcatcactt tattggtaat gttgagtcat aaggcagtta 120 caaaaggacc cccatggtga ccaaacaatg ttggactatg gcttagcttc tgtaatagga 180 tgaccgactc tcccgtcaca aagagctgag aaaggaaaga gaaaaccaca gtgatgacaa 240 agtacgacaa atggctgtgc tgtgaagtac cagaaagccc caatttttgg tgtatgtcgc 300 ccacttttcc tccaccagag actaagatgt catcccatag ctaagagaac ttagagggag 360 aaacagggcc gcacgtcatc ctccacttca ggtgaatttg tcactgcaag tggtgcaggg 420 atgttacaac caagccgcgg acaactcatg agtagggccg agattcctgg tactgtggag 480 aggcgcagag catatgaaca cctcaaacag atgattgtca cagggtggga aatccttcca 540 tgaaacagac acttgagaca tagtggcaaa gacattaagg ccttccattt ctttgcctac 600 accgnttaaa ggcactgata ccaacaacag aa 632 36 646 DNA Homo sapiens misc_feature Incyte ID No 71010168V1 36 cttcctagga gaatttcttt cttagctctc ccctcatgat agacacaggg ccaatggcag 60 gcccagcttc ccccggcagg agccactcct gtctcccacg ttccccacac agagctgtgg 120 tgactcaacc cacgtgtcag gaacaggacc gaacaagcgt atgtataaac gcagaccagg 180 atcatcactt tattggtaat gttgagtcat aaggcagtta caaaaggacc cccatggtga 240 ccaaacaatg ttggactatg gcttagcttc tgtaatagga tgaccgactc tcccgtcaca 300 aagagctgag aaaggaaaga gaaaaccaca gtgatgacaa agtacgacaa atggctgtgc 360 tggtgaagta ccagaaagcc ccaatttttg gtgtatgtcg cccacttttc ctccaccaga 420 gactaagatg tcatcccata gctaagagaa cttagaggga gaaacagggc cgcacgtcat 480 cctccacttc aggtgaattt gtcactgcaa gtggtgcagg gatgttacaa ccaagccgcg 540 gacaactcat gagtagggcc gagattcctg gtactgtgga gaggcgcaga gcatatgaac 600 acctcaaaca gatgatgtcc cagggtggga aatccttcca tgaaac 646 37 498 DNA Homo sapiens misc_feature Incyte ID No 70090181V1 37 agctttcacc tccagctttt tccacatcgg gattcacagg caatttagca ccccagccat 60 ggtttacaat acaggatgtt cagaacaatg aaggaagatg ggagccaccg agatccccga 120 gacccacaca cacactctaa tactgacgcg agagtgagct tcctgccacc accgctgtgg 180 gacccgaaaa tgttctctgt gtgatgtgta acggcttttc ctttgtcttt tatgtttcat 240 ggcagaacta cttaggagcg tggcatcgcc agtcagacat gcagtgtgtg gatgcattag 300 acaagcatca attgcctctg acttgaaaaa ccgtagagag cagatcaaaa tgactgtctg 360 gtttatctct gtcccattct gtgcccttcc tgacaagctg tcagaacaaa aactaattaa 420 aataattact aggatgtact gggccatgag ctcttcctag gagaaattct ttcttagctc 480 tcccctcatg atagacac 498 38 572 DNA Homo sapiens misc_feature Incyte ID No 6833928H1 38 cttgtcagga agggcacaga atgggacaga gataaaccag acagtcattt gatctgctct 60 ctacggtttt tcaagtcaga ggcaattgat gcttgtctaa tgcatccaca cactgcatgt 120 ctgactggcg atgccacgct cctaagtagt tctgccatga aacataaaag acaaaggaaa 180 agccgttaca catcacacag agaacatttt cgggtcccac agcggtggtg gcaggaagct 240 cactctcgcg tcagtattag agtgtgtgtg tgggtctcgg ggatctcggt ggctcccatc 300 ttccttcatt gttctgaaca tcctgtattg taaaccatgg ctggggtgct aaagtgcctg 360 tgaatcccga tgtggaaaaa gctggaggtg aaagctcagc ataccatgta tttactttaa 420 aaacagaaaa aaagacatgt atggatatgt ctattttttt tttatgggca catggtattt 480 ttgtgtggac ttgtttttag aaatgatgtg tccacacacg tacccgtgtc tcttctgcat 540 ttctgtgtca tggctctggt tcttaatcac gt 572 39 550 DNA Homo sapiens misc_feature Incyte ID No 70089663V1 39 gctcataagt agttctgcca tgaaacataa aagacaaagg aaaagccgtt acacatcaca 60 cagagaacat tttcgggtcc cacagcggtg gtggcaggaa gctcactctc gcgtcagtat 120 tagagtgtgt gtgtgggtct cggggatctc ggtggctccc atcttccttc attgttctga 180 acatcctgta ttgtaaacca tggctggggt gctaaagtgc ctgtgaatcc cgatgtggaa 240 aaagctggag gtgaaagctc agcataccat gtatttactt taaaaacaga aaaaaagaca 300 tgtatggata tgtctatttt ttttttattg gcacattgta tttttgtgtt gacttgtttt 360 tagaaatgat gtgtccacac acgtacccgt gtctcttctg catttctgtg tcatggttct 420 gtttcttaat cacgtgcggc ggtgtctaag tggtgttacc agtgtacgcg cagtgacctt 480 ggatgacagt ggctcttgct cacagcctcc cctgagctgt gagacacagc tttctctgta 540 catatgcaac 550 40 514 DNA Rattus norvegicus misc_feature Incyte ID No 702231139H1 40 aagcaatttt caaatgccag tcaattggat ttcgttaaaa cacgaaaatc aaaaagcatg 60 gatttaggta tagctgacga gaactaaact caatacagtg gatgaccaga gagcaggctc 120 ccccaataga gatgtgggac cctccttggg tctgaagaaa tccagctctt tagaaagtct 180 gcagacggct gttgctgagg tgaccctgaa tgggaacatt cctttccacc gcccacggcc 240 acgaatcatc cgaggaaggg gctgcaacga gagcttcaga gccgccattg acaagtccta 300 cgataagccc atggtggatg acgacgacga aggcatggag accttggaag aagacacaga 360 agaaagttca aggtcaggga gggagtccgt gtccacgtcc agtgatcagc cttcctattc 420 tctggagaga caaatgaatg gagacccaga gaagagggac aaggcagaga agaaaaagga 480 caaagccgga aaggataaga agaaagaccg agag 514 41 544 DNA Rattus norvegicus misc_feature Incyte ID No 700273304F6 41 cctgaatggg aacattcctt tccaccgccc acggccacga atcatccgag gaaggggctg 60 caacgagagc ttcagagccg ccattgacaa gtcctacgat aagcccatgg tggatgacga 120 cgacgaaggc atggagacct tggaagaaga cacagaagaa agttcaaggt cagggaggga 180 gtccgtgtcc acgtccagtg atcagccttc ctattctctg gagagacaaa tgaatggaga 240 cccagagaag agggacaagg cagagaagaa aaaggacaaa gccggaaagg ataagaagaa 300 agaccgagag aaggagaagg ataaactgaa agccaagaag gggatgctga aaggcttggg 360 ggacatgttc agcctggcca aactgaagcc ggagaagaga tgaacagcat gccagactca 420 aactgtcttg gacagcacaa gttgcacaat tgttttttaa aagcacggtg tctgggctgt 480 ggctcagtct agagtgcctg cctggtgtac acaaagccgt gggctcaatc cccagcaccc 540 tata 544 42 272 DNA Rattus norvegicus misc_feature Incyte ID No 700330856H1 42 tagattcagc gggcaagtcc caggaggaag ttgtttccct gttgagaagc accaagatgg 60 aggggaccgt gagccttctg gtctttcgtc aagaagaggc tttccagcca agggaaatga 120 atgccgaacc cagccagatg cagagtccaa aagaaacgaa agccgaagac gaggacattg 180 ttctcacacc tgacggtacc agggagtttc tgactttcga agttccactg aatgactcag 240 ggtctgcagg gcttggtgtc agcgtcaagg gg 272 43 300 DNA Rattus norvegicus misc_feature Incyte ID No 700273304H1 43 actgaatggg aacattcctt tccaccgccc acggccacga atcatccgag gaaggggctg 60 caacgagagc ttcagagccg ccattgacaa gtcctacgat aagcccatgg tggatgacga 120 cgacgaaggc atggagacct tggaagaaga cacagaagaa agttcaaggt cagggaggga 180 gtccgtgtcc acgtccagtg atcagccttc ctattctctg gagagacaaa tgaatggaga 240 cccagagaag agggacaagg cagagaagaa aaaggacaaa gccggaaagg ataagaagaa 300 44 300 DNA Rattus norvegicus misc_feature Incyte ID No 701517518H1 44 caggatctca cactccctct acagtgggat cgaggggctg gatgagtctc ccaccaggaa 60 tgccgcactc agcaggataa tgggtaaatg ccagctctcc ccaaccgtga acatgccaca 120 tgatgacact gtcatgattg aagatgacag gctgcctgtg ctccctcctc acctctctga 180 ccagtcctcc tccagctccc atgatgacgt gggattcata atgacagaag caggcacgtg 240 ggccaaggct accatcagtg actcagccga ctgctcattg actccagatg ttgatccggt 300 45 544 DNA Rattus norvegicus misc_feature Incyte ID No 701834089T1 45 aaacctgagt nnccttnaca acccaaagta aatttattgt ttggatttta aaaaaacttt 60 ctttgagaca cgtttcgtgt atcccaggct ggcctcgaac actacgtatg caggatgacc 120 ttgaacttcn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180 nnnnnnnnnn ntatagggtg ctggggattg agcccacggc tttgtgtaca ccaggcaggc 240 actctagact gagccacagc ccagacaccg tgcttttaaa aaacaattgt gcaacttgtg 300 ctgtccaaga cagtttgagt ctggcatgct gttcatctct tctccggctt cagtttggcc 360 aggctgaaca tgtcccccaa gcctttcagc atccccttct tggctttcag tttatccttc 420 tccttctctc ggtctttctt cttatccttt ccggctttgt cctttttctt ctctgccttg 480 tccctcttct ctgggtctcc attcatttgt ctctccagag aataggaagg ctgatcactg 540 gacg 544 46 196 DNA Rattus norvegicus misc_feature Incyte ID No 701480437H1 46 ctctctctct ctcatccttg actgactaac ttctttgctt tattgccaga caaagcagga 60 agaatgccag ctctgtatca caggattcct gggaacagaa ctacgcccct ggtgaaggct 120 tccagagtgc caaggagaac cccaggtatt ccagttacca gggctccagg aacggctatc 180 taggtggcca tggctt 196 47 273 DNA Rattus norvegicus misc_feature Incyte ID No 701190235H1 47 gcagatgtaa cgagttgcgg tctcctggga gccccgctgc acccgatctg cccatacaaa 60 cagagttgga tgacagacaa cgcaggatct cacactccct ctacagtggg atcgatgggc 120 tggatgagtc tcccaccagg aatgccgcac tcagcaggat aatgggtaaa tgccagctct 180 ccccaaccgt gaacatgcta catgatgaca ctgtcatgat tgaagatgac aggctgcctg 240 tgctcactcc tcacctctct gaccagtcct cct 273 48 248 DNA Rattus norvegicus misc_feature Incyte ID No 700939688H1 48 cagagaagag ggacaaggca gagaagaaaa aggacaaagc cggaaaggat aagaagaaag 60 accgagagaa ggagaaggat aaactgaaag ccaagaaggg gatgctgaaa ggcttggggg 120 acatgttcag cctggccaaa ctgaagccgg agaagagatg aacagcatgc cagactcaaa 180 ctgtcttgga cagcacaagt tgcacaattg ttttttaaaa gcacggtgtc tgggctgtgg 240 ctcagtct 248 49 351 DNA Rattus norvegicus misc_feature Incyte ID No 700939688F6 49 cagagaagag ggacaaggca gagaagaaaa aggacaaagc cggaaaggat aagaagaaag 60 accgagagaa ggagaaggat aaactgaaag ccaagaaggg gatgctgaaa ggcttggggg 120 acatgttcag cctggccaaa ctgaagccgg agaagagatg aacagcatgc cagactcaaa 180 ctgtcttgga cagcacaagt tgcacaattg ttttttaaaa gcacggtgtc tgggctgtgg 240 ctcagtctag aagatgcctg cctggctgta cacaagagcc agtggagctc aagtccccag 300 acagccctat agaaccagcg tgtggtagac acatgcnctg tcatcccagc a 351 50 571 DNA Rattus norvegicus misc_feature Incyte ID No 702582937T1 50 cactttagca acccagcctt ggtttacaat acaggatgtt cagaccaaca gatgaacggc 60 gggaacacgg agggcctcgt gccacaggca tgcacggaga atgggactcc cggtgctcag 120 agggacatcg acaggtcctc gagtgggatg gctctccttc tgtttgtgaa taaacagcag 180 agtcactcag taatgttggc ctcgtcaggt cgggacatgg tatgaggata taggagacca 240 aatcctgact gcaacctcaa aagctgtgtt gaggttgatt ctcagaatcc caagtgactg 300 acctttttcc ttgatcccac tctgtgcctc ccttgacaac ctacggtgac acgaagtaaa 360 gtaaggactg gatagaccgg cctaagctcc tccagagagt cttccctcag actcctatct 420 ccttcctcgg ggtgcgtaca catgggccac tcccatgccc cttgttcccg agtgtcatga 480 gtgactgaaa ctgaacgcat gtacttatag aaccactgac tagaaatcgg ctgtagatat 540 gggtgggtgg agttataaag ggcagttgga a 571 51 694 DNA Rattus norvegicus misc_feature Incyte ID No 700299037F6 51 ctctagggtg gtagtgaaga agctaagcca taggccagtg ccgttggttc tggggggtga 60 gggtaacttt ccaactgcct tataactcca ccacccatat cacagcgatt ctagtcagtg 120 tttataagta catgcgttca gtttcagtca ctcatgacac tcgggaacaa ggggcatggg 180 agtggcccat gtgtacgcac cccgaggaag gagataggag ctgagggaag actctctgga 240 ggagcttagg ccggtctatc cagtccttac tttacttcgt gtcaccgtag gttgtcaagg 300 gaggcacaga gtgggacaag gaaaaaggtc agtcacttgg gattctgaga atcaacctca 360 acacagcttt tgaggttgca gtcaggattg gtctcctata tcctcatacc atgtcccgac 420 ctgacgaggc caacattact gagtgactct gctgtttatt cacaaacaga aggagagcca 480 tcccactcga ggacctgtcg atgtccctct gagcaccggg agtcccattc tccgtgcatg 540 cctgtggcac gaggcctccg tgttcccgcc gttcatctgt tggtctgaac atcctgtatg 600 taaaccaagg ctgggttgct aaagtgcctg agaatctcga tataaaaaac aaaaaacaaa 660 aaaatccttg gggcaaaagc tcagagtacc atgt 694 52 110 DNA Rattus norvegicus misc_feature Incyte ID No 701246488H1 52 gattgaagat gacaggctgc ctgtgctccc tcctgacctc tctgaccagg cgtcctccag 60 ctcccatgat gacgtgggat tcataatgac agaagcaggc acgtgggcca 110 53 578 DNA Canis familiaris misc_feature Incyte ID No 702759912H1 53 atgaaagtga ccgtgtgctt cgggcggacc cgggtggtcg tgccgtgcgg ggacgggcac 60 atgaaagttt tcagcctcat ccagcaagcg gtgacccgct accggaaggc catcgccaag 120 gatccaaact actggataca ggtgcaccga cttgaacatg gagatggagg aatactagac 180 cttgatgaca ccctctgtga tgtagcagat gataaagaca gactggtagc agtgtttgat 240 gagcaagatc cacatcatgg aggtgatggc accagtgcca gctccacagg tacccagagt 300 ccagagatat ttggcagtga gcttggcacc aacaatgttt cagcctttca gccttatcaa 360 gctacaagtg aaattgaggt cacaccttca gttcttcgtg caaatatgcc tcttcatgtc 420 cgacgaagca gtgacccggc tttaattggc ctttcaactt ccatcagtga cactaatttt 480 ccttctgaag agccttcacg gaagaacccc acacgttggt caacaacagc tggctttctg 540 aagcaaaaca ctgctggcag ccctaatact gtgacaaa 578 54 293 DNA Mus musculus misc_feature Incyte ID No 700112340H1 54 gggcatttga ctgagatgtc ccaaaggtgc ctattggaag agcattatga tccaaactac 60 tggatacagg tgcatcgctt ggagcatgga gatggaggga ttctagacct ggatgacatc 120 ctctgtgacg ttgctgatga caaagacaga ctggtagcag tatttgatga acaggatccc 180 caccatggag gagatggtac cagcgccagc tccacgggaa cccagagtcc agagatattc 240 ggcagtgagc tgggcaccaa caatgtttct gcttttcagc cttatcaagc cac 293 55 233 DNA Mus musculus misc_feature Incyte ID No 700827810H1 55 cgcggccggc atcgcagagt ggtcggcact cggtgtccgt ggaggttcaa gtacaacggc 60 agcgccagga ggagcgagag agcttccagc aggcccagcg ccagtacagc tcactgccaa 120 gacaaagcag gaagaatgcc agctccatat cacaggattc ctgggaacag aagagtgaag 180 aaatctttgg gcaagtatgg ccctagcagt gtagaagaca ccacaggaag tgg 233 56 222 DNA Mus musculus misc_feature Incyte ID No 700109331H1 56 gggcatctca aatgcaagga aaactaatct ttttgccaaa ttgacacttt gtaaatttat 60 tttctctatt gctaaaaata aaatagacat gtgtttggga ccctgagtct catcccgaag 120 catccgaacc ttactcaaag aatcatggag attgtactca ctacctaaat ccatgctttt 180 tgattttcgt gttttaacga aatccaattg actggcatct ga 222 57 369 DNA Homo sapiens misc_feature Incyte ID No g6661750 57 aaggggcgct gccgcgagcc tccgggcctc agggtgttcc ggggagcggc gccccgggtc 60 tctgggccca cccgccccgg gcgtcctccg agagtggggg ctgcgcccgc ggggtcagac 120 acctgttcgg cccggcccgg cgtggtcgcc gggggccagg atgaaagtga ccgtgtgctt 180 cggcaggacg ggcatcgtgg tgccctgcaa ggagggccag ctgcgcgtcg gcgagctcac 240 ccagcaggcg ctgcagcggt acctgaagac ccgggagaag ggtcctggtt actgggtgaa 300 gattcatcac ttagaatata cagatggagg aatcctggat ccagatgatg tcttggcaga 360 tgttgttga 369 58 511 DNA Homo sapiens misc_feature Incyte ID No GNN.g10801482_004.edit 58 gccccggccg cacatggttc gaggccgagg ctgcaatgag agctttagag cagccattga 60 caaatcctac gatggacctg aagaaataga agctgacggt ctgtctgata agagctctca 120 ctctggccaa ggagctctga attgtgagtc tgcccctcag gggaattcgg agctagagga 180 catggaaaat aaagccagga aagtcaaaaa aacgaaagag aaggagaaga aaaaggaaaa 240 gggcaaattg aaagtcaagg agaaaaagcg caaagaggag aatgaagatc cagaaaggaa 300 aataaagaag aagggcttcg gcgccatgct gaggtatggg cctgctttga aggcaaagtt 360 ggttctcatt ttgtctctcc tgaaaaaagc gcacgctttt cctcgtcttc agccaaatgc 420 atacggctct caattctgtg ctcgttctct ttctgcagag gcagaggagc tttttgggga 480 aagttacagt gatgacagga cactgtctta a 511 59 591 DNA Homo sapiens misc_feature Incyte ID No g6993427 59 ccagttttat ccttttttct atcagtctta tcacctttct cttggtttcc attcatttgt 60 ctctccagag agtgggaagg ctgatcactg gctgtggata cagactctct ccctgatctt 120 gaactttctt ctgtgtcttc ttccaaggtc tccatgcctt catcatcatc atctaccacg 180 ggtttatcat aagatttgtc gatggcagct ctgaagctct cattgcatcc cctgcctctg 240 attatccgcg gccgtggacg atggaaagga atatccccat tcaaagtcac ctcggcaact 300 gcggtctgca gactctccaa cgagcttgac ttcttcagac ccagggaagg acccacatct 360 ctgctgggag aacctgcttt ctggtcatcc actgtattga gtttagtctc gtcagctata 420 cctaaatcca tgctttttga ttttcgtgtt ttaacgaaat ccaattgact ggcatctgaa 480 aattgctttg tgcgtttttc tgacatactc tgacgtccaa atccttctcg ttgaaaagca 540 agaactggat caacatctgg actcaaagag cagtcgggtg aatcactgat t 591 60 389 DNA Homo sapiens misc_feature Incyte ID No g5529915 60 ttttttttca gttttatcct tttttctatc agtcttatca cctttctctt ggtttccatt 60 catttgtctc tccagagagt gggaaggctg atcactggct gtggatacag actctctccc 120 tgatcttgaa ctttcttctg tgtcttcttc caaggtctcc atgccttcat catcatcatc 180 taccgcgggt ttatcataag atttgtcgat ggcagctctg aagctctcat tgcatcccct 240 gcctctgatt atccgcggcc gtggacgatg gaaaggaata tccccattca aagtcacctc 300 ggcaactgcg gtctgcagac tctccaacga gcttgacttc ttcagaccca gggaaggacc 360 cacatctctg ctgggagaac ctgctttct 389 61 367 DNA Homo sapiens misc_feature Incyte ID No g1733437 61 ggaaaccaag agaaaggtga taagactgat agaaaaaagg ataaaactgg aaaagaaaag 60 aagaaagata gagataagga gaaggataaa atgaaagcca agaagggaat gctgaagggc 120 ttgggagaca tgttcaggtt tggcaaacat cgaaaagatg acaagattga gaaaacgggt 180 aaaataaaaa tacaggaatc ctttacatca gaagaggaga ggatacgaat gaagcaggag 240 caggagagga ttcaagccaa aactcgagaa tttagggaac cgacaagctc gagagcgtga 300 ctatgctgaa attcaagatt ttcatcggac atttggctgt gatgatgagt taatgtatgg 360 gggagtt 367 62 1337 PRT Rattus norvegicus misc_feature Incyte ID No g3868778 62 Met Lys Val Thr Val Cys Phe Gly Arg Thr Arg Val Val Val Pro 1 5 10 15 Cys Gly Asp Gly Arg Met Lys Val Phe Ser Leu Ile Gln Gln Ala 20 25 30 Val Thr Arg Tyr Arg Lys Ala Val Ala Lys Asp Pro Asn Tyr Trp 35 40 45 Ile Gln Val His Arg Leu Glu His Gly Asp Gly Gly Ile Leu Asp 50 55 60 Leu Asp Asp Ile Leu Cys Asp Val Ala Asp Asp Lys Asp Arg Leu 65 70 75 Val Ala Val Phe Asp Glu Gln Asp Pro His His Gly Gly Asp Gly 80 85 90 Thr Ser Ala Ser Ser Thr Gly Thr Gln Ser Pro Glu Ile Phe Gly 95 100 105 Ser Glu Leu Gly Thr Asn Asn Val Ser Ala Phe Arg Pro Tyr Gln 110 115 120 Thr Thr Ser Glu Ile Glu Val Thr Pro Ser Val Leu Arg Ala Asn 125 130 135 Met Pro Leu His Val Arg Arg Ser Ser Asp Pro Ala Leu Thr Gly 140 145 150 Leu Ser Thr Ser Val Ser Asp Asn Asn Phe Ser Ser Glu Glu Pro 155 160 165 Ser Arg Lys Asn Pro Thr Arg Trp Ser Thr Thr Ala Gly Phe Leu 170 175 180 Lys Gln Asn Thr Thr Gly Ser Pro Lys Thr Cys Asp Arg Lys Lys 185 190 195 Asp Glu Asn Tyr Arg Ser Leu Pro Arg Asp Pro Ser Ser Trp Ser 200 205 210 Asn Gln Phe Gln Arg Asp Asn Ala Arg Ser Ser Leu Ser Ala Ser 215 220 225 His Pro Met Val Asp Arg Trp Leu Glu Lys Gln Glu Gln Asp Glu 230 235 240 Glu Gly Thr Glu Glu Asp Ser Ser Arg Val Glu Pro Val Gly His 245 250 255 Ala Asp Thr Gly Leu Glu Asn Met Pro Asn Phe Ser Leu Asp Asp 260 265 270 Met Val Lys Leu Val Gln Val Pro Asn Asp Gly Gly Pro Leu Gly 275 280 285 Ile His Val Val Pro Phe Ser Ala Arg Gly Gly Arg Thr Leu Gly 290 295 300 Leu Leu Val Lys Arg Leu Glu Lys Gly Gly Lys Ala Glu Gln Glu 305 310 315 Asn Leu Phe His Glu Asn Asp Cys Ile Val Arg Ile Asn Asp Gly 320 325 330 Asp Leu Arg Asn Arg Arg Phe Glu Gln Ala Gln His Met Phe Arg 335 340 345 Gln Ala Met Arg Ala Arg Val Ile Trp Phe His Val Val Pro Ala 350 355 360 Ala Asn Lys Glu Gln Tyr Glu Gln Leu Ser Gln Arg Glu Met Asn 365 370 375 Asn Tyr Ser Pro Gly Arg Phe Ser Pro Asp Ser His Cys Val Ala 380 385 390 Asn Arg Ser Val Ala Asn Asn Ala Pro Gln Ala Leu Pro Arg Ala 395 400 405 Pro Arg Leu Ser Gln Pro Pro Glu Gln Leu Asp Ala His Pro Arg 410 415 420 Leu Pro His Ser Ala His Ala Ser Thr Lys Pro Pro Thr Ala Pro 425 430 435 Ala Leu Ala Pro Pro Asn Val Leu Ser Thr Ser Val Gly Ser Val 440 445 450 Tyr Asn Thr Lys Arg Val Gly Lys Arg Leu Asn Ile Gln Leu Lys 455 460 465 Lys Gly Thr Glu Gly Leu Gly Phe Ser Ile Thr Ser Arg Asp Val 470 475 480 Thr Ile Gly Gly Ser Ala Pro Ile Tyr Val Lys Asn Ile Leu Pro 485 490 495 Arg Gly Ala Ala Ile Gln Asp Gly Arg Leu Lys Ala Gly Asp Arg 500 505 510 Leu Ile Glu Val Asn Gly Val Asp Leu Ala Gly Lys Ser Gln Glu 515 520 525 Glu Val Val Ser Leu Leu Arg Ser Thr Lys Met Glu Gly Thr Val 530 535 540 Ser Leu Leu Val Phe Arg Gln Glu Glu Ala Phe His Pro Arg Glu 545 550 555 Met Asn Ala Glu Pro Ser Gln Met Gln Ser Pro Lys Glu Thr Lys 560 565 570 Ala Glu Asp Glu Asp Ile Val Leu Thr Pro Asp Gly Thr Arg Glu 575 580 585 Phe Leu Thr Phe Glu Val Pro Leu Asn Asp Ser Gly Ser Ala Gly 590 595 600 Leu Gly Val Ser Val Lys Gly Asn Arg Ser Lys Glu Asn His Ala 605 610 615 Asp Leu Gly Ile Phe Val Lys Ser Ile Ile Asn Gly Gly Ala Ala 620 625 630 Ser Lys Asp Gly Arg Leu Arg Val Asn Asp Gln Leu Ile Ala Val 635 640 645 Asn Gly Glu Ser Leu Leu Gly Lys Ala Asn Gln Glu Ala Met Glu 650 655 660 Thr Leu Arg Arg Ser Met Ser Thr Glu Gly Asn Lys Arg Gly Met 665 670 675 Ile Gln Leu Ile Val Ala Arg Arg Ile Ser Arg Cys Asn Glu Leu 680 685 690 Arg Ser Pro Gly Ser Pro Ala Ala Pro Glu Leu Pro Ile Glu Thr 695 700 705 Glu Leu Asp Asp Arg Glu Arg Arg Ile Ser His Ser Leu Tyr Ser 710 715 720 Gly Ile Glu Gly Leu Asp Glu Ser Pro Thr Arg Asn Ala Ala Leu 725 730 735 Ser Arg Ile Met Gly Glu Ser Gly Lys Cys Gln Leu Ser Pro Thr 740 745 750 Val Asn Met Pro His Asp Asp Thr Val Met Ile Glu Asp Asp Arg 755 760 765 Leu Pro Val Leu Pro Pro His Leu Ser Asp Gln Ser Ser Ser Ser 770 775 780 Ser His Asp Asp Val Gly Phe Ile Met Thr Glu Ala Gly Thr Trp 785 790 795 Ala Lys Ala Thr Ile Ser Asp Ser Ala Asp Cys Ser Leu Ser Pro 800 805 810 Asp Val Asp Pro Val Leu Ala Phe Gln Arg Glu Gly Phe Gly Arg 815 820 825 Gln Ser Met Ser Glu Lys Arg Thr Lys Gln Phe Ser Asn Ala Ser 830 835 840 Gln Leu Asp Phe Val Lys Thr Arg Lys Ser Lys Ser Met Asp Leu 845 850 855 Gly Ile Ala Asp Glu Thr Lys Leu Asn Thr Val Asp Asp Gln Arg 860 865 870 Ala Gly Ser Pro Asn Arg Asp Val Gly Pro Ser Leu Gly Leu Lys 875 880 885 Lys Ser Ser Ser Leu Glu Ser Leu Gln Thr Ala Val Ala Glu Val 890 895 900 Thr Leu Asn Gly Asn Ile Pro Phe His Arg Pro Arg Pro Arg Ile 905 910 915 Ile Arg Gly Arg Gly Cys Asn Glu Ser Phe Arg Ala Ala Ile Asp 920 925 930 Lys Ser Tyr Asp Lys Pro Met Val Asp Asp Asp Asp Glu Gly Met 935 940 945 Glu Thr Leu Glu Glu Asp Thr Glu Glu Ser Ser Arg Ser Gly Arg 950 955 960 Glu Ser Val Ser Thr Ser Ser Asp Gln Pro Ser Tyr Ser Leu Glu 965 970 975 Arg Gln Met Asn Gly Asp Pro Glu Lys Arg Asp Lys Ala Glu Lys 980 985 990 Lys Lys Asp Lys Ala Gly Lys Asp Lys Lys Lys Asp Arg Glu Lys 995 1000 1005 Glu Lys Asp Lys Leu Lys Ala Lys Lys Gly Met Leu Lys Gly Leu 1010 1015 1020 Gly Asp Met Phe Arg Phe Gly Lys His Arg Lys Asp Asp Lys Met 1025 1030 1035 Glu Lys Met Gly Arg Ile Lys Ile Gln Asp Ser Phe Thr Ser Glu 1040 1045 1050 Glu Asp Arg Val Arg Met Lys Glu Glu Gln Glu Arg Ile Gln Ala 1055 1060 1065 Lys Thr Arg Glu Phe Arg Glu Arg Gln Ala Arg Glu Arg Asp Tyr 1070 1075 1080 Ala Glu Ile Gln Asp Phe His Arg Thr Phe Gly Cys Asp Asp Glu 1085 1090 1095 Leu Leu Tyr Gly Gly Met Ser Ser Tyr Asp Gly Cys Leu Ala Leu 1100 1105 1110 Asn Ala Arg Pro Gln Ser Pro Arg Glu Gly His Leu Met Asp Thr 1115 1120 1125 Leu Tyr Ala Gln Val Lys Lys Pro Arg Ser Ser Lys Pro Gly Asp 1130 1135 1140 Ser Asn Arg Ser Thr Pro Ser Asn His Asp Arg Ile Gln Arg Leu 1145 1150 1155 Arg Gln Glu Phe Gln Gln Ala Lys Gln Asp Glu Asp Val Glu Asp 1160 1165 1170 Arg Arg Arg Thr Tyr Ser Phe Glu Gln Ser Trp Ser Ser Ser Arg 1175 1180 1185 Pro Ala Ser Gln Ser Gly Arg His Ser Val Ser Val Glu Val Gln 1190 1195 1200 Val Gln Arg Gln Arg Gln Glu Glu Arg Glu Ser Phe Gln Gln Ala 1205 1210 1215 Gln Arg Gln Tyr Ser Ser Leu Pro Arg Gln Ser Arg Lys Asn Ala 1220 1225 1230 Ser Ser Val Ser Gln Asp Ser Trp Glu Gln Asn Tyr Ala Pro Gly 1235 1240 1245 Glu Gly Phe Gln Ser Ala Lys Glu Asn Pro Arg Tyr Ser Ser Tyr 1250 1255 1260 Gln Gly Ser Arg Asn Gly Tyr Leu Gly Gly His Gly Phe Asn Ala 1265 1270 1275 Arg Val Met Leu Glu Thr Gln Glu Leu Leu Arg Gln Glu Gln Arg 1280 1285 1290 Arg Lys Glu Gln Gln Leu Lys Lys Gln Pro Pro Ala Asp Gly Val 1295 1300 1305 Arg Gly Pro Phe Arg Gln Asp Val Pro Pro Ser Pro Ser Gln Val 1310 1315 1320 Ala Arg Leu Asn Arg Leu Gln Thr Pro Glu Lys Gly Arg Pro Phe 1325 1330 1335 Tyr Ser 63 1266 PRT Homo sapiens misc_feature Incyte ID No g8037915 63 Met Lys Val Thr Val Cys Phe Gly Arg Thr Arg Val Val Val Pro 1 5 10 15 Cys Gly Asp Gly His Met Lys Val Phe Ser Leu Ile Gln Gln Ala 20 25 30 Val Thr Arg Tyr Arg Lys Ala Ile Ala Lys Asp Pro Asn Tyr Trp 35 40 45 Ile Gln Val His Arg Leu Glu His Gly Asp Gly Gly Ile Leu Asp 50 55 60 Leu Asp Asp Ile Leu Cys Asp Val Ala Asp Asp Lys Asp Arg Leu 65 70 75 Val Ala Val Phe Asp Glu Gln Asp Pro His His Gly Gly Asp Gly 80 85 90 Thr Ser Ala Ser Ser Thr Gly Thr Gln Ser Pro Glu Ile Phe Gly 95 100 105 Ser Glu Leu Gly Thr Asn Asn Val Ser Ala Phe Gln Pro Tyr Gln 110 115 120 Ala Thr Ser Glu Ile Glu Val Thr Pro Ser Val Leu Arg Ala Asn 125 130 135 Met Pro Leu His Val Arg Arg Ser Ser Asp Pro Ala Leu Ile Gly 140 145 150 Leu Ser Thr Ser Val Ser Asp Ser Asn Phe Ser Ser Glu Glu Pro 155 160 165 Ser Arg Lys Asn Pro Thr Arg Trp Ser Thr Thr Ala Gly Phe Leu 170 175 180 Lys Gln Asn Thr Ala Gly Ser Pro Lys Thr Cys Asp Arg Lys Asp 185 190 195 Glu Asp Gly Thr Glu Glu Asp Asn Ser Arg Val Glu Pro Val Gly 200 205 210 His Ala Asp Thr Gly Leu Glu His Ile Pro Asn Phe Ser Leu Asp 215 220 225 Asp Met Val Lys Leu Val Glu Val Pro Asn Asp Gly Gly Pro Leu 230 235 240 Gly Ile His Val Val Pro Phe Ser Ala Arg Gly Gly Arg Thr Leu 245 250 255 Gly Leu Leu Val Lys Arg Leu Glu Lys Gly Gly Lys Ala Glu His 260 265 270 Glu Asn Leu Phe Arg Glu Asn Asp Cys Ile Val Arg Ile Asn Asp 275 280 285 Gly Asp Leu Arg Asn Arg Arg Phe Glu Gln Ala Gln His Met Phe 290 295 300 Arg Gln Ala Met Arg Thr Pro Ile Ile Trp Phe His Val Val Pro 305 310 315 Ala Ala Asn Lys Glu Gln Tyr Glu Gln Leu Ser Gln Ser Glu Lys 320 325 330 Asn Asn Tyr Tyr Ser Ser Arg Phe Ser Pro Asp Ser Gln Tyr Ile 335 340 345 Asp Asn Arg Ser Val Asn Ser Ala Gly Leu His Thr Val Gln Arg 350 355 360 Ala Pro Arg Leu Asn His Pro Pro Glu Gln Ile Asp Ser His Ser 365 370 375 Arg Leu Pro His Ser Ala His Pro Ser Gly Lys Pro Pro Ser Ala 380 385 390 Pro Ala Ser Ala Pro Gln Asn Val Phe Ser Thr Thr Val Ser Ser 395 400 405 Gly Tyr Asn Thr Lys Lys Ile Gly Lys Arg Leu Asn Ile Gln Leu 410 415 420 Lys Lys Gly Thr Glu Gly Leu Gly Phe Ser Ile Thr Ser Arg Asp 425 430 435 Val Thr Ile Gly Gly Ser Ala Pro Ile Tyr Val Lys Asn Ile Leu 440 445 450 Pro Arg Gly Ala Ala Ile Gln Asp Gly Arg Leu Lys Ala Gly Asp 455 460 465 Arg Leu Ile Glu Val Asn Gly Val Asp Leu Val Gly Lys Ser Gln 470 475 480 Glu Glu Val Val Ser Leu Leu Arg Ser Thr Lys Met Glu Gly Thr 485 490 495 Val Ser Leu Leu Val Phe Arg Gln Glu Asp Ala Phe His Pro Arg 500 505 510 Glu Leu Lys Ala Glu Asp Glu Asp Ile Val Leu Thr Pro Asp Gly 515 520 525 Thr Arg Glu Phe Leu Thr Phe Glu Val Pro Leu Asn Asp Ser Gly 530 535 540 Ser Ala Gly Leu Gly Val Ser Val Lys Gly Asn Arg Ser Lys Glu 545 550 555 Asn His Ala Asp Leu Gly Ile Phe Val Lys Ser Ile Ile Asn Gly 560 565 570 Gly Ala Ala Ser Lys Asp Gly Arg Leu Arg Val Asn Asp Gln Leu 575 580 585 Ile Ala Val Asn Gly Glu Ser Leu Leu Gly Lys Thr Asn Gln Asp 590 595 600 Ala Met Glu Thr Leu Arg Arg Ser Met Ser Thr Glu Gly Asn Lys 605 610 615 Arg Gly Met Ile Gln Leu Ile Val Ala Arg Arg Ile Ser Lys Cys 620 625 630 Asn Glu Leu Lys Ser Pro Gly Ser Pro Pro Gly Pro Glu Leu Pro 635 640 645 Ile Glu Thr Ala Leu Asp Asp Arg Glu Arg Arg Ile Ser His Ser 650 655 660 Leu Tyr Ser Gly Ile Glu Gly Leu Asp Glu Ser Pro Ser Arg Asn 665 670 675 Ala Ala Leu Ser Arg Ile Met Gly Lys Tyr Gln Leu Ser Pro Thr 680 685 690 Val Asn Met Pro Gln Asp Asp Thr Val Ile Ile Glu Asp Asp Arg 695 700 705 Leu Pro Val Leu Pro Pro His Leu Ser Asp Gln Ser Ser Ser Ser 710 715 720 Ser His Asp Asp Val Gly Phe Val Thr Ala Asp Ala Gly Thr Trp 725 730 735 Ala Lys Ala Ala Ile Ser Asp Ser Ala Asp Cys Ser Leu Ser Pro 740 745 750 Asp Val Asp Pro Val Leu Ala Phe Gln Arg Glu Gly Phe Gly Arg 755 760 765 Gln Ile Ala Asp Glu Thr Lys Leu Asn Thr Val Asp Asp Gln Lys 770 775 780 Ala Gly Ser Pro Ser Arg Asp Val Gly Pro Ser Leu Gly Leu Lys 785 790 795 Lys Ser Ser Ser Leu Glu Ser Leu Gln Thr Ala Val Ala Glu Val 800 805 810 Thr Leu Asn Gly Asp Ile Pro Phe His Arg Pro Arg Pro Arg Ile 815 820 825 Ile Arg Gly Arg Gly Cys Asn Glu Ser Phe Arg Ala Ala Ile Asp 830 835 840 Lys Ser Tyr Asp Lys Pro Ala Val Asp Asp Asp Asp Glu Gly Met 845 850 855 Glu Thr Leu Glu Glu Asp Thr Glu Glu Ser Ser Arg Ser Gly Arg 860 865 870 Glu Ser Val Ser Thr Ala Ser Asp Gln Pro Ser His Ser Leu Glu 875 880 885 Arg Gln Met Asn Gly Asn Gln Glu Lys Gly Asp Lys Thr Asp Arg 890 895 900 Lys Lys Asp Lys Thr Gly Lys Glu Lys Lys Lys Asp Arg Asp Lys 905 910 915 Glu Lys Asp Lys Met Lys Ala Lys Lys Gly Met Leu Lys Gly Leu 920 925 930 Gly Asp Met Phe Arg Phe Gly Lys His Arg Lys Asp Asp Lys Ile 935 940 945 Glu Lys Thr Gly Lys Ile Lys Ile Gln Glu Ser Phe Thr Ser Glu 950 955 960 Glu Glu Arg Ile Arg Met Lys Gln Glu Gln Glu Arg Ile Gln Ala 965 970 975 Lys Thr Arg Glu Phe Arg Glu Arg Gln Ala Arg Glu Arg Asp Tyr 980 985 990 Ala Glu Ile Gln Asp Phe His Arg Thr Phe Gly Cys Asp Asp Glu 995 1000 1005 Leu Met Tyr Gly Gly Val Ser Ser Tyr Glu Gly Ser Met Ala Leu 1010 1015 1020 Asn Ala Arg Pro Gln Ser Pro Arg Glu Gly His Met Met Asp Ala 1025 1030 1035 Leu Tyr Ala Gln Val Lys Lys Pro Arg Asn Ser Lys Pro Ser Pro 1040 1045 1050 Val Asp Ser Asn Arg Ser Thr Pro Ser Asn His Asp Arg Ile Gln 1055 1060 1065 Arg Leu Arg Gln Glu Phe Gln Gln Ala Lys Gln Asp Glu Asp Val 1070 1075 1080 Glu Asp Arg Arg Arg Thr Tyr Ser Phe Glu Gln Pro Trp Pro Asn 1085 1090 1095 Ala Arg Pro Ala Thr Gln Ser Gly Arg His Ser Val Ser Val Glu 1100 1105 1110 Val Gln Met Gln Arg Gln Arg Gln Glu Glu Arg Glu Ser Ser Gln 1115 1120 1125 Gln Ala Gln Arg Gln Tyr Ser Ser Leu Pro Arg Gln Ser Arg Lys 1130 1135 1140 Asn Ala Ser Ser Val Ser Gln Asp Ser Trp Glu Gln Asn Tyr Ser 1145 1150 1155 Pro Gly Glu Gly Phe Gln Ser Ala Lys Glu Asn Pro Arg Tyr Ser 1160 1165 1170 Ser Tyr Gln Gly Ser Arg Asn Gly Tyr Leu Gly Gly His Gly Phe 1175 1180 1185 Asn Ala Arg Val Met Leu Glu Thr Gln Glu Leu Leu Arg Gln Glu 1190 1195 1200 Gln Arg Arg Lys Glu Gln Gln Met Lys Lys Gln Pro Pro Ser Glu 1205 1210 1215 Gly Pro Ser Asn Tyr Asp Ser Tyr Lys Lys Val Gln Asp Pro Ser 1220 1225 1230 Tyr Ala Pro Pro Lys Gly Pro Phe Arg Gln Asp Val Pro Pro Ser 1235 1240 1245 Pro Ser Gln Val Ala Arg Leu Asn Arg Leu Gln Thr Pro Glu Lys 1250 1255 1260 Gly Arg Pro Phe Tyr Ser 1265 

What is claimed is:
 1. An isolated cDNA encoding a protein having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
 2. An isolated cDNA encoding a protein having the amino acid sequence of SEQ ID NO:
 1. 3. An isolated cDNA encoding a protein having the amino acid sequence of SEQ ID NO:2.
 4. An isolated cDNA selected from: a) a nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:20 or the complement thereof; b) a fragment of SEQ ID NO:3 selected from SEQ ID NOs:4-11 or the complement thereof or a fragment of SEQ ID NO:20 selected from SEQ ID NOs:21-39 or the complement thereof; and c) a variant of SEQ ID NO:3 selected from SEQ ID NOs:12-19 or a variant of SEQ ID NO:20 selected from SEQ ID NOs:40-56.
 5. A composition comprising the cDNA or the complement of the cDNA of claim
 1. 6. A vector comprising the cDNA of claim
 1. 7. A host cell comprising the vector of claim
 6. 8. A method for using a cDNA to produce a protein, the method comprising: a) culturing the host cell of claim 7 under conditions for protein expression; and b) recovering the protein from the host cell culture.
 9. A method for using a cDNA to detect expression of a nucleic acid in a sample comprising: a) hybridizing the composition of claim 5 to nucleic acids of the sample, thereby forming hybridization complexes; and b) comparing hybridization complex formation with a standard, wherein the comparison indicates expression of the cDNA in the sample.
 10. The method of claim 9 further comprising amplifying the nucleic acids of the sample prior to hybridization.
 11. The method of claim 9 wherein the composition is attached to a substrate.
 12. The method of claim 9 wherein the cDNA is differentially expressed when compared with the standard and diagnostic of bladder transitional cell carcinoma.
 13. A method of using a cDNA to screen a plurality of molecules or compounds, the method comprising: a) combining the cDNA of claim 1 with a plurality of molecules or compounds under conditions to allow specific binding; and b) detecting specific binding, thereby identifying a molecule or compound which specifically binds the cDNA.
 14. The method of claim 13 wherein the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, transcription factors, repressors, and regulatory molecules.
 15. A purified protein or a portion thereof selected from: a) an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2; b) an antigenic epitope of SEQ ID NO: 1 or SEQ ID NO:2; and c) a biologically active portion of SEQ ID NO: 1 or SEQ ID NO:2.
 16. A composition comprising the protein of claim
 15. 17. A method for using a protein to screen a plurality of molecules or compounds to identify at least one ligand, the method comprising: a) combining the protein of claim 15 with the molecules or compounds under conditions to allow specific binding; and b) detecting specific binding, thereby identifying a ligand which specifically binds the protein.
 18. The method of claim 17 wherein the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs.
 19. A method of using a protein to prepare and purify antibodies comprising: a) immunizing a animal with the protein of claim 15 under conditions to elicit an antibody response; b) isolating animal antibodies; c) attaching the protein to a substrate; d) contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein; e) dissociating the antibodies from the protein, thereby obtaining purified antibodies.
 20. An antibody produced by the method of claim
 19. 21. A method for using an antibody to diagnose conditions or diseases associated with expression of a protein, the method comprising: a) combining the antibody of claim 20 with a sample, thereby forming antibody:protein complexes; and b) comparing complex formation with a standard, wherein the comparison indicates expression of the protein in the sample.
 22. The method of claim 21 wherein expression is diagnostic of bladder transitional cell carcinoma. 